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1.  Histological Transformation and Progression in Follicular Lymphoma: A Clonal Evolution Study 
PLoS Medicine  2016;13(12):e1002197.
Background
Follicular lymphoma (FL) is an indolent, yet incurable B cell malignancy. A subset of patients experience an increased mortality rate driven by two distinct clinical end points: histological transformation and early progression after immunochemotherapy. The nature of tumor clonal dynamics leading to these clinical end points is poorly understood, and previously determined genetic alterations do not explain the majority of transformed cases or accurately predict early progressive disease. We contend that detailed knowledge of the expansion patterns of specific cell populations plus their associated mutations would provide insight into therapeutic strategies and disease biology over the time course of FL clinical histories.
Methods and Findings
Using a combination of whole genome sequencing, targeted deep sequencing, and digital droplet PCR on matched diagnostic and relapse specimens, we deciphered the constituent clonal populations in 15 transformation cases and 6 progression cases, and measured the change in clonal population abundance over time. We observed widely divergent patterns of clonal dynamics in transformed cases relative to progressed cases. Transformation specimens were generally composed of clones that were rare or absent in diagnostic specimens, consistent with dramatic clonal expansions that came to dominate the transformation specimens. This pattern was independent of time to transformation and treatment modality. By contrast, early progression specimens were composed of clones that were already present in the diagnostic specimens and exhibited only moderate clonal dynamics, even in the presence of immunochemotherapy. Analysis of somatic mutations impacting 94 genes was undertaken in an extension cohort consisting of 395 samples from 277 patients in order to decipher disrupted biology in the two clinical end points. We found 12 genes that were more commonly mutated in transformed samples than in the preceding FL tumors, including TP53, B2M, CCND3, GNA13, S1PR2, and P2RY8. Moreover, ten genes were more commonly mutated in diagnostic specimens of patients with early progression, including TP53, BTG1, MKI67, and XBP1.
Conclusions
Our results illuminate contrasting modes of evolution shaping the clinical histories of transformation and progression. They have implications for interpretation of evolutionary dynamics in the context of treatment-induced selective pressures, and indicate that transformation and progression will require different clinical management strategies.
Sohrab Shah and colleagues explore the evolutionary histories that shape clinical and transformation dynamics in follicular lymphoma
Author Summary
Why Was This Study Done?
Follicular lymphoma (FL) is a largely incurable malignancy in which early progression and transformation have consistently been linked to lymphoma-related mortality.
We contended that detailed characterization of clonal dynamics would reveal fundamental biological properties with implications for future patient management strategies relating to both transformation and progression.
We also sought to identify recurrent gene mutations associated with transformation and/or early progression in a large patient cohort.
What Did the Researchers Do and Find?
Using whole genome sequencing, deep allelic sampling by amplicon sequencing, and digital droplet PCR, we found dramatic clonal expansions in transformed disease, whereby dominant clones in transformation samples emerged from extremely low prevalence clones or from clones that were not detected in the diagnostic samples.
The dynamics of disease progression during treatment in the absence of transformation showed markedly different characteristics, with much of the clonal architecture preserved from diagnostic to relapse specimens.
Targeted capture-based sequencing in a large extension cohort then established genetic variants associated with transformation and early progression in the broader patient population.
What Do These Findings Mean?
Taken together, our findings illuminate previously undescribed patterns of clonal expansion underpinning FL clinical histories suggesting that contrasting management strategies will be necessary across the FL patient population.
We uncovered novel associations of gene mutations with early progression that could inform future prognostic assay development.
doi:10.1371/journal.pmed.1002197
PMCID: PMC5154502  PMID: 27959929
2.  The binding selectivity of vonoprazan (TAK-438) to the gastric H+,K+−ATPase 
Summary
Background
The gastric H+,K+-ATPase is the preferred target for acid suppression. Until recently, the only drugs that effectively inhibited this ATPase were the proton pump inhibitors (PPIs). PPIs are acid-activated prodrugs that require acid protection. Once acid activated, PPIs bind to cysteines of the ATPase, resulting in covalent, long-lasting inhibition. The short plasma half-life of PPIs and continual de novo synthesis of the H+,K+-ATPase result in difficulty controlling nighttime acid secretion. A new alternative to PPIs is the pyrrolo-pyridine, vonoprazan (TAK-438), a potassium-competitive acid blocker (PCAB) that does not require acid protection. In contrast to other PCABs, vonoprazan has a long duration of action, resulting in 24 hour control of acid secretion, a high pKa of 9.37 and high affinity (Ki = 3.0 ηM).
Aim
To determine binding selectivity of vonoprazan for the gastric H+,K+-ATPase and to explain its slow dissociation.
Methods
Gastric gland and parietal cell binding of vonoprazan was determined radiometrically. Molecular modeling explained the slow dissociation of vonoprazan from the H+,K+-ATPase.
Results
Vonoprazan binds selectively to the parietal cell, independent of acid secretion. Vonoprazan binds in a luminal vestibule between the surfaces of membrane helices 4, 5 and 6. Exit of the drug to the lumen is hindered by asp137 and asn138 in the loop between TM1 and TM2, which presents an electrostatic barrier to movement of the sulfonyl group of vonoprazan. This may explain its slow dissociation from the H+,K+-ATPase and long lasting inhibition.
Conclusions
The binding model provides a template for design of novel PCABs.
doi:10.1111/apt.13414
PMCID: PMC4626316  PMID: 26423447
H+,K+-ATPase; TAK-438; vonoprazan; parietal cell; molecular modeling
3.  Technical Report: Correlation Between the Repair of Cartilage and Subchondral Bone in an Osteochondral Defect Using Bilayered, Biodegradable Hydrogel Composites 
Tissue Engineering. Part C, Methods  2015;21(12):1216-1225.
The present work investigated correlations between cartilage and subchondral bone repair, facilitated by a growth factor-delivering scaffold, in a rabbit osteochondral defect model. Histological scoring indices and microcomputed tomography morphological parameters were used to evaluate cartilage and bone repair, respectively, at 6 and 12 weeks. Correlation analysis revealed significant associations between specific cartilage indices and subchondral bone parameters that varied with location in the defect (cortical vs. trabecular region), time point (6 vs. 12 weeks), and experimental group (insulin-like growth factor-1 only, bone morphogenetic protein-2 only, or both growth factors). In particular, significant correlations consistently existed between cartilage surface regularity and bone quantity parameters. Overall, correlation analysis between cartilage and bone repair provided a fuller understanding of osteochondral repair and can help drive informed studies for future osteochondral regeneration strategies.
doi:10.1089/ten.tec.2015.0117
PMCID: PMC4663641  PMID: 26177155
4.  Risk factors for reading disability in rolandic epilepsy families 
Epilepsy & behavior : E&B  2015;53:174-179.
OBJECTIVE
The high prevalence and impact of neurodevelopmental comorbidities in childhood epilepsy are now well known, as are the increased risks and familial aggregation of reading disability (RD) and speech sound disorder (SSD) in rolandic epilepsy (RE). The risk factors for RD in the general population include male sex, SSD and ADHD but it is not known if these are the same in RE or whether there is a contributory role of seizure and treatment related variables.
METHODS
An observational study of 108 RE probands (age range 3.6–22 years) and their 159 siblings (age range 1–29 years; 83 with EEG data) singly ascertained in the US or UK through an affected RE proband. We used a nested case-control design, multiple logistic regression and generalized estimating equations to test the hypothesis of association between RD and seizure variables or antiepileptic drug treatment in RE; we also assessed an association between EEG focal sharp waves and RD in siblings.
RESULTS
RD was reported in 42% of probands and 22% of siblings. Among probands, RD was strongly associated with a history of SSD (OR 9.64, 95% CI: 2.45–37.21), ADHD symptoms (OR 10.31, 95% CI: 2.15–49.44), and male sex (OR 3.62, 95% CI: 1.11–11.75), but not with seizure or treatment variables. Among siblings, RD was independently associated only with SSD (OR 4.30, 95%CI: 1.42–13.0) and not with the presence of interictal EEG focal sharp waves.
SIGNIFICANCE
The principal risk factors for RD in RE are SSD, ADHD and male sex, the same risk factors as for RD without epilepsy. Seizure or treatment variables do not appear to be important risk factors for RD in RE probands, and there was no evidence to support interictal EEG focal sharp waves as a risk factor for RD in siblings. Future studies should focus on the precise neuropsychological characterisation of RD in RE families, and on the effectiveness of standard oral-language and reading interventions.
doi:10.1016/j.yebeh.2015.10.016
PMCID: PMC4719157  PMID: 26580214
epilepsy; neurodevelopment; comorbidity; aetiology; reading
5.  FOXO responses to Porphyromonas gingivalis in epithelial cells 
Cellular microbiology  2015;17(11):1605-1617.
Summary
Porphyromonas gingivalis is a prominent periodontal, and emerging systemic, pathogen that redirects host cell signalling pathways and modulates innate immune responses. In this study, we show that P. gingivalis infection induces the dephosphorylation and activation of forkhead box-O (FOXO)1, 3 and 4 in gingival epithelial cells. In addition, immunofluorescence showed that FOXO1 accumulated in the nucleus of P. gingivalis-infected cells. Quantitative reverse transcription PCR demonstrated that transcription of genes involved in protection against oxidative stress (Cat, Sod2, Prdx3), inflammatory responses (IL1β) and anti-apoptosis (Bcl-6) was induced by P. gingivalis, while small-interfering RNA (siRNA)-mediated knockdown of FOXO1 suppressed the transcriptional activation of these genes. P. gingivalis-induced secretion of interleukin (IL)-1β and inhibition of apoptosis were also impeded by FOXO1 knockdown. Neutralization of reactive oxygen species (ROS) by N-acetyl-l-cysteine blocked the activation of FOXO1 by P. gingivalis and concomitantly suppressed the activation of oxidative stress responses, anti-apoptosis programmes and IL-β production. Inhibition of c-Jun-N-terminal kinase (JNK) either pharmacologically or by siRNA, reduced FOXO1 activation and downstream FOXO1-dependent gene regulation in response to P. gingivalis. The results indicate that P. gingivalis-induced ROS activate FOXO transcription factors through JNK signalling, and that FOXO1 controls oxidative stress responses, inflammatory cytokine production and cell survival. These data position FOXO as an important signalling node in the epithelial cell–P. gingivalis interaction, with particular relevance to cell fate and dysbiotic host responses.
doi:10.1111/cmi.12459
PMCID: PMC4624012  PMID: 25958948
6.  STAT2 Is a Pervasive Cytokine Regulator due to Its Inhibition of STAT1 in Multiple Signaling Pathways 
PLoS Biology  2016;14(10):e2000117.
STAT2 is the quintessential transcription factor for type 1 interferons (IFNs), where it functions as a heterodimer with STAT1. However, the human and murine STAT2-deficient phenotypes suggest important additional and currently unidentified type 1 IFN-independent activities. Here, we show that STAT2 constitutively bound to STAT1, but not STAT3, via a conserved interface. While this interaction was irrelevant for type 1 interferon signaling and STAT1 activation, it precluded the nuclear translocation specifically of STAT1 in response to IFN-γ, interleukin-6 (IL-6), and IL-27. This is explained by the dimerization between activated STAT1 and unphosphorylated STAT2, whereby the semiphosphorylated dimers adopted a conformation incapable of importin-α binding. This, in turn, substantially attenuated cardinal IFN-γ responses, including MHC expression, senescence, and antiparasitic immunity, and shifted the transcriptional output of IL-27 from STAT1 to STAT3. Our results uncover STAT2 as a pervasive cytokine regulator due to its inhibition of STAT1 in multiple signaling pathways and provide an understanding of the type 1 interferon-independent activities of this protein.
Author Summary
For more than a quarter century, we have known that STAT1 and STAT2 are essential for the classic host immune defense system against viral infections known as the type 1 interferon response. While STAT1 has since been assigned multiple additional roles, STAT2 is thought to function exclusively as the principal partner of STAT1 in the type 1 interferon system. However, patients and animals that are deficient in STAT2 show a surprisingly varied and sometimes subtle phenotype not fully accounted for by the known functions of this protein. Our investigations reveal an entirely novel facet of STAT2 action, namely as an innate inhibitor of STAT1 in its multiple biological roles. We identify the molecular mechanism of STAT1 inhibition and generate a novel biological tool with which we can dissociate STAT2’s activating and inhibitory effects on STAT1. We use this tool to show that STAT2 has major roles beyond antiviral protection, for example, in regulating cell proliferation and immune cell functions, as well as in killing intracellular parasites. These findings considerably expand our knowledge of STAT2 biology and necessitate a reassessment of regulatory mechanisms central to innate immunity and the therapeutic use of interferons.
doi:10.1371/journal.pbio.2000117
PMCID: PMC5079630  PMID: 27780205
7.  Atomistic modelling of scattering data in the Collaborative Computational Project for Small Angle Scattering (CCP-SAS)1  
Journal of Applied Crystallography  2016;49(Pt 6):1861-1875.
The CCP-SAS project is currently developing software for the atomistic and coarse-grained molecular modelling of X-ray and neutron small-angle scattering data. Its computational framework is described, alongside applications in biology and soft matter.
The capabilities of current computer simulations provide a unique opportunity to model small-angle scattering (SAS) data at the atomistic level, and to include other structural constraints ranging from molecular and atomistic energetics to crystallography, electron microscopy and NMR. This extends the capabilities of solution scattering and provides deeper insights into the physics and chemistry of the systems studied. Realizing this potential, however, requires integrating the experimental data with a new generation of modelling software. To achieve this, the CCP-SAS collaboration (http://www.ccpsas.org/) is developing open-source, high-throughput and user-friendly software for the atomistic and coarse-grained molecular modelling of scattering data. Robust state-of-the-art molecular simulation engines and molecular dynamics and Monte Carlo force fields provide constraints to the solution structure inferred from the small-angle scattering data, which incorporates the known physical chemistry of the system. The implementation of this software suite involves a tiered approach in which GenApp provides the deployment infrastructure for running applications on both standard and high-performance computing hardware, and SASSIE provides a workflow framework into which modules can be plugged to prepare structures, carry out simulations, calculate theoretical scattering data and compare results with experimental data. GenApp produces the accessible web-based front end termed SASSIE-web, and GenApp and SASSIE also make community SAS codes available. Applications are illustrated by case studies: (i) inter-domain flexibility in two- to six-domain proteins as exemplified by HIV-1 Gag, MASP and ubiquitin; (ii) the hinge conformation in human IgG2 and IgA1 antibodies; (iii) the complex formed between a hexameric protein Hfq and mRNA; and (iv) synthetic ‘bottlebrush’ polymers.
doi:10.1107/S160057671601517X
PMCID: PMC5139988  PMID: 27980506
molecular dynamics (MD); molecular modelling; scattering curve fits; small-angle-neutron scattering (SANS); small-angle-X-ray scattering (SAXS)
8.  Biodegradable, Phosphate-containing, Dual-Gelling Macromers for Cellular Delivery in Bone Tissue Engineering 
Biomaterials  2015;67:286-296.
Injectable, biodegradable, dual-gelling macromer solutions were used to encapsulate mesenchymal stem cells (MSCs) within stable hydrogels when elevated to physiologic temperature. Pendant phosphate groups were incorporated in the N-isopropyl acrylamide-based macromers to improve biointegration and facilitate hydrogel degradation. The MSCs were shown to survive the encapsulation process, and live cells were detected within the hydrogels for up to 28 days in vitro. Cell-laden hydrogels were shown to undergo significant mineralization in osteogenic medium. Cell-laden and acellular hydrogels were implanted into a critical-size rat cranial defect for 4 and 12 weeks. Both cell-laden and acellular hydrogels were shown to degrade in vivo and help to facilitate bone growth into the defect. Improved bone bridging of the defect was seen with the incorporation of cells, as well as with higher phosphate content of the macromer. Furthermore, direct bone-to-hydrogel contact was observed in the majority of implants, which is not commonly seen in this model. The ability of these macromers to deliver stem cells while forming in situ and subsequently degrade while facilitating bone ingrowth into the defect makes this class of macromers a promising material for craniofacial bone tissue engineering.
doi:10.1016/j.biomaterials.2015.07.016
PMCID: PMC4550499  PMID: 26232878
hydrogel; poly(N-isopropyl acrylamide); monoacryloxyethyl phosphate; rat cranial defect; mesenchymal stem cell encapsulation
9.  Colloidal bismuth subcitrate (CBS) impedes proton entry into Helicobacter pylori and increases the efficacy of growth dependent antibiotics 
Background
Successful eradication of Helicobacter pylori is becoming more difficult, mainly due to emerging antibiotic resistance. Treatment regimens containing bismuth have increased efficacy, but the mechanism is unknown. H. pylori is a neutralophile adapted to survive the acidic gastric environment via acid acclimation, but demonstrates more robust growth at neutral pH. Many antibiotics used to treat H. pylori rely on bacterial growth.
Aim
To investigate the mechanism of increased efficacy of bismuth-containing H. pylori treatment regimens.
Methods
RNAseq and qPCR, urease activity in permeabilized and intact bacteria, internal pH, and membrane potential were measured with and without colloidal bismuth subcitrate (CBS). Bacterial survival was assessed with CBS and/or ampicillin.
Results
Genes involved with metabolism and growth were upregulated in the presence of CBS at acidic pH. Urease activity of permeabilized H. pylori at pH 7.4 and 4.5 decreased in the presence of CBS, but intact urease activity only decreased at acidic pH. The fall in cytoplasmic pH with external acidification was diminished by CBS. The increase in membrane potential in response to urea addition at acidic medium pH was unaffected by CBS. The impact of CBS and ampicillin on H. pylori survival was greater than either agent alone.
Conclusions
Bismuth is not acting directly on urease or the urea channel. CBS impedes proton entry into the bacteria, leading to a decrease in the expected fall in cytoplasmic pH. With cytoplasmic pH remaining within range for increased metabolic activity of a neutralophile, the efficacy of growth dependent antibiotics is augmented.
doi:10.1111/apt.13346
PMCID: PMC4558396  PMID: 26238858
Helicobacter pylori; bismuth; cytoplasmic pH
10.  Prognostic Significance of Diffuse Large B-Cell Lymphoma Cell of Origin Determined by Digital Gene Expression in Formalin-Fixed Paraffin-Embedded Tissue Biopsies 
Journal of Clinical Oncology  2015;33(26):2848-2856.
Purpose
To evaluate the prognostic impact of cell-of-origin (COO) subgroups, assigned using the recently described gene expression–based Lymph2Cx assay in comparison with International Prognostic Index (IPI) score and MYC/BCL2 coexpression status (dual expressers).
Patients and Methods
Reproducibility of COO assignment using the Lymph2Cx assay was tested employing repeated sampling within tumor biopsies and changes in reagent lots. The assay was then applied to pretreatment formalin-fixed paraffin-embedded tissue (FFPET) biopsies from 344 patients with de novo diffuse large B-cell lymphoma (DLBCL) uniformly treated with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) at the British Columbia Cancer Agency. MYC and BCL2 protein expression was assessed using immunohistochemistry on tissue microarrays.
Results
The Lymph2Cx assay provided concordant COO calls in 96% of 49 repeatedly sampled tumor biopsies and in 100% of 83 FFPET biopsies tested across reagent lots. Critically, no frank misclassification (activated B-cell–like DLBCL to germinal center B-cell–like DLBCL or vice versa) was observed. Patients with activated B-cell–like DLBCL had significantly inferior outcomes compared with patients with germinal center B-cell–like DLBCL (log-rank P < .001 for time to progression, progression-free survival, disease-specific survival, and overall survival). In pairwise multivariable analyses, COO was associated with outcomes independent of IPI score and MYC/BCL2 immunohistochemistry. The prognostic significance of COO was particularly evident in patients with intermediate IPI scores and the non–MYC-positive/BCL2-positive subgroup (log-rank P < .001 for time to progression).
Conclusion
Assignment of DLBCL COO by the Lymph2Cx assay using FFPET biopsies identifies patient groups with significantly different outcomes after R-CHOP, independent of IPI score and MYC/BCL2 dual expression.
doi:10.1200/JCO.2014.60.2383
PMCID: PMC4554747  PMID: 26240231
11.  When a Module is not a Domain – the Case of the REJ Module and the Redefinition of the Architecture of Polycystin-1 
The Biochemical journal  2011;435(3):651-660.
Synopsis
The extracellular region of a group of cell surface receptors known as the polycystic kidney disease 1 family, comprising amongst others polycystin-1, has been controversially described as containing four fibronectin type III (FNIII) domains or one REJ module in the same portion of polypeptide. Stimulated by recent atomic force microscopy work we re-examined the similarity of these four domains with a FNIII sequence profile showing the evolutionary relationship. Two of the predicted domains could be expressed in bacteria and refolded to give protein suitable for biophysical study and one of these expressed solubly. Circular dichroism spectroscopy showed that both domains contain a significant amount of β-sheet, in good agreement with theoretical predictions. Confirmation of independent folding as a domain is obtained from highly cooperative thermal and urea unfolding curves. Excellent dispersion of peaks in the high field region of one dimensional NMR spectra confirms the presence of a hydrophobic core. Analytical ultracentrifugation and analytical gel filtration agree very well with the narrow linewidths in the NMR spectra that at least one of the domains is monomeric. Based on this combined theoretical and experimental analysis we show that the extracellular portion of polycystin-1 does indeed contain β-sheet domains, very likely fibronectin type III, and that consequently the REJ module is not a single domain.
doi:10.1042/BJ20101810
PMCID: PMC4979573  PMID: 21314639
12.  Tension on JAM-A activates RhoA via GEF-H1 and p115 RhoGEF 
Molecular Biology of the Cell  2016;27(9):1420-1430.
Forces on JAM-A activate RhoA to increase cell stiffness. Activation of RhoA requires GEF-H1 and p115 RhoGEF activation downstream of FAK/ERK and Src family kinases, respectively.
Junctional adhesion molecule A (JAM-A) is a broadly expressed adhesion molecule that regulates cell–cell contacts and facilitates leukocyte transendothelial migration. The latter occurs through interactions with the integrin LFA-1. Although we understand much about JAM-A, little is known regarding the protein’s role in mechanotransduction or as a modulator of RhoA signaling. We found that tension imposed on JAM-A activates RhoA, which leads to increased cell stiffness. Activation of RhoA in this system depends on PI3K-mediated activation of GEF-H1 and p115 RhoGEF. These two GEFs are further regulated by FAK/ERK and Src family kinases, respectively. Finally, we show that phosphorylation of JAM-A at Ser-284 is required for RhoA activation in response to tension. These data demonstrate a direct role of JAM-A in mechanosignaling and control of RhoA and implicate Src family kinases in the regulation of p115 RhoGEF.
doi:10.1091/mbc.E15-12-0833
PMCID: PMC4850030  PMID: 26985018
13.  Suppression of PGC-1α is critical for reprogramming oxidative metabolism in renal cell carcinoma 
Cell reports  2015;12(1):116-127.
Summary
Long believed to be a byproduct of malignant transformation, reprogramming of cellular metabolism is now recognized as a driving force in tumorigenesis. In clear cell renal cell carcinoma (ccRCC) frequent activation of HIF-signaling induces a metabolic switch that promotes tumorigenesis. Here we demonstrate that PGC-1α, a central regulator of energy metabolism, is suppressed in VHL-deficient ccRCC by a HIF/Dec1-dependent mechanism. In VHL wild type cells, PGC-1α suppression leads to decreased expression of the mitochondrial transcription factor Tfam and impaired mitochondrial respiration. Conversely, PGC-1α expression in VHL-deficient cells restores mitochondrial function and induces oxidative stress. ccRCC cells expressing PGC-1α exhibit impaired tumor growth and enhanced sensitivity to cytotoxic therapies. In patients, low levels of PGC-1α expression are associated with poor outcome. These studies demonstrate that suppression of PGC-1α recapitulates key metabolic phenotypes of ccRCC and highlight the potential of targeting PGC-1α expression as a therapeutic modality for the treatment of ccRCC.
doi:10.1016/j.celrep.2015.06.006
PMCID: PMC4518559  PMID: 26119730
14.  Optimal responses in disease activity scores to treatment in rheumatoid arthritis: Is a DAS28 reduction of >1.2 sufficient? 
Background
The overall benefit of intensive treatment strategies in rheumatoid arthritis (RA) remains uncertain. We explored how reductions in disability and improvements in quality of life scores are affected by alternative assessments of reductions in disease activity scores for 28 joints (DAS28) in two trials of intensive treatment strategies in active RA.
Methods
One trial (CARDERA) studied 467 patients with early active RA receiving 24 months of methotrexate monotherapy or steroid and disease-modifying anti-rheumatic drug (DMARD) combinations. The other trial (TACIT) studied 205 patients with established active RA; they received 12 months of treatment with DMARD combinations or biologic agents. We compared changes in the health assessment questionnaire (HAQ) and Euroqol-5D (EQ5D) at trial endpoints in European League Against Rheumatism (EULAR) good and moderate EULAR responders in patients in whom complete endpoint data were available.
Results
In the CARDERA trial 98 patients (26 %) were good EULAR responders and 160 (32 %) were EULAR moderate responders; comparable data in TACIT were 66 (35 %) and 86 (46 %) patients. The magnitude of change in the HAQ and EQ5D was greater in both trials in EULAR good responders than in EULAR moderate responders. HAQ scores had a difference in of –0.49 (95 % CI –0.66, –0.32) in the CARDERA and –0.31 (95 % CI –0.47, –0.13) in the TACIT trial. With the EQ5D comparable differences were 0.12 (95 % CI 0.04, 0.19) and 0.15 (95 % CI 0.05, 0.25). Both exceeded minimum clinically important differences in HAQ and EQ5D scores.
Conclusions
We conclude that achieving a good EULAR response with DMARDs and biologic agents in active RA results in substantially improved mean HAQ and EQ5D scores. Patients who achieve such responses should continue on treatment. However, continuing such treatment strategies is more challenging when only a moderate EULAR response is achieved. In these patients evidence of additional clinically important benefits in measures such as the HAQ should also be sought.
doi:10.1186/s13075-016-1028-8
PMCID: PMC4910246  PMID: 27312203
Rheumatoid arthritis; DAS28; Response criteria
15.  Development of an On-animal Separation-based Sensor for Monitoring Drug Metabolism in Freely Roaming Sheep 
The Analyst  2015;140(11):3820-3829.
The development of an on-animal separation-based sensor that can be employed for monitoring drug metabolism in a freely roaming sheep is described. The system consists of microdialysis sampling coupled directly to microchip electrophoresis with electrochemical detection (MD-ME-EC). Separations were accomplished using an all-glass chip with integrated platinum working and reference electrodes. Discrete samples from the microdialysis flow were introduced into the electrophoresis chip using a flow-gated injection approach. Electrochemical detection was accomplished in-channel using a two-electrode isolated potentiostat. Nitrite was separated by microchip electrophoresis using reverse polarity and a run buffer consisting of 50 mM phosphate at pH 7.4. The entire system was under telemetry control. The system was first tested with rats to monitor the production of nitrite following introduction of nitroglycerin into the subdermal tissue using a linear probe. The data acquired using the on-line MD-ME-EC system was compared to that obtained off-line analysis by liquid chromatography with electrochemical detection (LC-EC), using a second microdialysis probe implanted parallel to the first probe in the same animal. The MD-ME-EC device was then used on-animal to monitor the subdermal metabolism of nitroglycerin in sheep. The ultimate goal is to use this device to simultaneously monitor drug metabolism and behavior in a freely roaming animal.
doi:10.1039/c4an01928h
PMCID: PMC4437826  PMID: 25697221
microchip electrophoresis; microdialysis; electrochemical detection; nitrite; sheep; nitroglycerin; skin
16.  A systematic review of randomised controlled trials in rheumatoid arthritis: the reporting and handling of missing data in composite outcomes 
Trials  2016;17:272.
Background
Most reported outcome measures in rheumatoid arthritis (RA) trials are composite, whose components comprise single measures that are combined into one outcome. The aims of this review were to assess the range of missing data rates in primary composite outcomes and to document the current practice for handling and reporting missing data in published RA trials compared to the Consolidated Standards of Reporting Trials (CONSORT) recommendations.
Methods
A systematic search for randomised controlled trials was conducted for RA trials published between 2008 and 2013 in four rheumatology and four high impact general medical journals.
Results
A total of 51 trials with a composite primary outcome were identified, of which 38 (75 %) used the binary American College of Rheumatology responder index and 13 (25 %) used the Disease Activity Score for 28 joints (DAS28). Forty-four trials (86 %) reported on an intention-to-treat analysis population, while 7 trials (14 %) analysed according to a modified intention-to-treat population. Missing data rates for the primary composite outcome ranged from 2–53 % and were above 30 % in 9 trials, 20–30 % in 11 trials, 10–20 % in 18 trials and below 10 % in 13 trials. Thirty-eight trials (75 %) used non-responder imputation and 10 (20 %) used last observation carried forward to impute missing composite outcome data at the primary time point. The rate of dropout was on average 61 % times higher in the placebo group compared to the treatment group in the 34 placebo controlled trials (relative rate 1.61, 95 % CI: 1.29, 2.02). Thirty-seven trials (73 %) did not report the use of sensitivity analyses to assess the handling of missing data in the primary analysis as recommended by CONSORT guidelines.
Conclusions
This review highlights an improvement in rheumatology trial practice since the revision of CONSORT guidelines, in terms of power calculation and participant’s flow diagram. However, there is a need to improve the handling and reporting of missing composite outcome data and their components in RA trials. In particular, sensitivity analyses need to be more widely used in RA trials because imputation is widespread and generally uses single imputation methods, and in this area the missing data rates are commonly differentially higher in the placebo group.
Electronic supplementary material
The online version of this article (doi:10.1186/s13063-016-1402-5) contains supplementary material, which is available to authorized users.
doi:10.1186/s13063-016-1402-5
PMCID: PMC4890523  PMID: 27255212
RA; Composite outcomes; Missing data; Imputation; Sensitivity analysis
17.  Porphyromonas gingivalis RagB is a pro-inflammatory STAT4 agonist 
Molecular oral microbiology  2015;30(3):242-252.
Summary
Periodontal diseases are semi-ubiquitous and caused by chronic, plaque-induced inflammation. The 55kDa immunodominant RagB outer membrane protein of Porphyromonas gingivalis, a keystone periodontal pathogen, has been proposed to facilitate nutrient transport. However, potential interactions between RagB and the innate response have not been examined. We determined that RagB exposure led to the differential and dose-related expression of multiple genes encoding pro-inflammatory mediators (IL-1α, IL-1β, IL-6, IL-8 and CCL2; all, p < 0.05) in primary human monocytes and to the secretion of TNF and IL-8, but not IFN-γ or IL-12. RagB was shown to be a TLR2 and TLR4 agonist that activated STAT4 and NFκB signaling. In keeping, a ΔragB mutant similarly exhibited reduced inflammatory capacity which was rescued by ragB complementation. These results suggest that RagB elicits a major pro-inflammatory response in primary human monocytes and, thus, could play an important role in the etiology of periodontitis and systemic sequelae.
doi:10.1111/omi.12089
PMCID: PMC4624316  PMID: 25418117
inflammation; monocyte; periodontitis; Porphyromonas gingivalis; STAT4; TLR
18.  Increased infection with key periodontal pathogens during gestational diabetes mellitus 
Aim
Gestational diabetes mellitus (GDM), gingivitis, infection with specific periodontal pathogens and systemic inflammation each increase the risk for poor pregnancy outcome. We set out to monitor the interactions of gingivitis and GDM with respect to oral infection and the systemic inflammatory burden.
Materials and Methods
Four case–control groups (n = 117) were recruited, (1) No gingivitis, No GDM (n = 27); (2) Gingivitis, No GDM (n = 31); (3) No gingivitis, GDM (n = 21); and (4) Gingivitis, GDM (n = 38). Oral infection with three key periodontal pathogens was determined by PCR. Systemic inflammation was determined by quantification of CRP by EIA.
Results
Gingivitis during pregnancy was associated with oral infection with Porphyromonas gingivalis, Filifactor alocis and Treponema denticola and combinations thereof (all p < 0.01). GDM was also associated with increased infection with individual and multiple oral pathogens (all p < 0.05). Gingivitis during pregnancy led to a 325% increase in systemic CRP (mean, 2495 versus 8116 ng/ml, p < 0.01).
Conclusions
Diabetes and gingivitis act in concert to increase risk biomarkers for poor pregnancy outcome.
doi:10.1111/jcpe.12418
PMCID: PMC4699310  PMID: 25959628
CRP; gestational diabetes; gingivitis; periodontopathogens; pregnancy
19.  Accelerometer‐determined physical activity, muscle mass, and leg strength in community‐dwelling older adults 
Abstract
Introduction
The aim of this study was to describe the relationship between accelerometer‐determined physical activity (PA), muscle mass, and lower‐limb strength in community‐dwelling older adults.
Methods
Six hundred thirty‐six community‐dwelling older adults (66 ± 7 years) were studied. Muscle mass was measured using dual‐energy x‐ray absorptiometry, whilst lower limb strength was measured via dynamometry. We measured minutes/day spent in sedentary, light, moderate, and vigorous intensity activity using Actigraph GT1M accelerometers.
Results
Participants spent a median of 583(Interquartile ratio (IQR) 522–646), 225(176–271), 27(12–45) and 0(0–0) min in sedentary, light, moderate, and vigorous activity, respectively. PA intensity was positively associated with both lean mass percentage and lower limb strength in a dose–response fashion. Sedentary activity was negatively associated with lean mass percentage, but not lower‐limb strength. There was a positive association between PA and appendicular lean mass in men only. There was an interaction between age and activity; as age increased, the magnitude of the association of PA with lean mass percentage decreased. Those who adhered to the Australian Department of Health PA guidelines (moderate/vigorous PA >/=150 min/week) had greater lean mass percentage, appendicular lean mass, and lower limb strength.
Conclusions
Using accelerometer technology, both the amount and intensity of accelerometer‐determined PA had an independent, dose–response relationship with lean mass percentage and lower limb strength, with the largest effect for vigorous activity. Time spent in sedentary activity was negatively associated with lean mass percentage, but was not associated with lower limb strength. The magnitude of the association between PA and lean mass percentage decreased with age, suggesting that PA programmes may need to be modified with increasing age.
doi:10.1002/jcsm.12065
PMCID: PMC4863829  PMID: 27239404
Physical activity; Accelerometer; Muscle mass; Strength
20.  Single-Patient Molecular Testing with NanoString nCounter Data Using a Reference-Based Strategy for Batch Effect Correction 
PLoS ONE  2016;11(4):e0153844.
A major weakness in many high-throughput genomic studies is the lack of consideration of a clinical environment where one patient at a time must be evaluated. We examined generalizable and platform-specific sources of variation from NanoString gene expression data on both ovarian cancer and Hodgkin lymphoma patients. A reference-based strategy, applicable to single-patient molecular testing is proposed for batch effect correction. The proposed protocol improved performance in an established Hodgkin lymphoma classifier, reducing batch-to-batch misclassification while retaining accuracy and precision. We suggest this strategy may facilitate development of NanoString and similar molecular assays by accelerating prospective validation and clinical uptake of relevant diagnostics.
doi:10.1371/journal.pone.0153844
PMCID: PMC4838303  PMID: 27096160
21.  Flexibility of KorA, a plasmid-encoded, global transcription regulator, in the presence and the absence of its operator 
Nucleic Acids Research  2016;44(10):4947-4956.
The IncP (Incompatibility group P) plasmids are important carriers in the spread of antibiotic resistance across Gram-negative bacteria. Gene expression in the IncP-1 plasmids is stringently controlled by a network of four global repressors, KorA, KorB, TrbA and KorC interacting cooperatively. Intriguingly, KorA and KorB can act as co-repressors at varying distances between their operators, even when they are moved to be on opposite sides of the DNA. KorA is a homodimer with the 101-amino acid subunits, folding into an N-terminal DNA-binding domain and a C-terminal dimerization domain. In this study, we have determined the structures of the free KorA repressor and two complexes each bound to a 20-bp palindromic DNA duplex containing its consensus operator sequence. Using a combination of X-ray crystallography, nuclear magnetic resonance spectroscopy, SAXS and molecular dynamics calculations, we show that the linker between the two domains is very flexible and the protein remains highly mobile in the presence of DNA. This flexibility allows the DNA-binding domains of the dimer to straddle the operator DNA on binding and is likely to be important in cooperative binding to KorB. Unexpectedly, the C-terminal domain of KorA is structurally similar to the dimerization domain of the tumour suppressor p53.
doi:10.1093/nar/gkw191
PMCID: PMC4889941  PMID: 27016739
22.  Registered report: IDH mutation impairs histone demethylation and results in a block to cell differentiation 
eLife  null;483:474-478.
The Reproducibility Project: Cancer Biology seeks to address growing concerns about reproducibility in scientific research by conducting replications of selected experiments from a number of high-profile papers in the field of cancer biology. The papers, which were published between 2010 and 2012, were selected on the basis of citations and Altmetric scores (Errington et al., 2014). This Registered Report describes the proposed replication plan of key experiments from “IDH mutation impairs histone demethylation and results in a block to cell differentiation” by Lu and colleagues, published in Nature in 2012 (Lu et al., 2012). The experiments that will be replicated are those reported in Figures 1B, 2A, 2B, 2D and 4D. Lu and colleagues demonstrated that expression of mutant forms of IDH1 or IDH2 caused global increases in histone methylation and increased levels of 2 hydroxyglutarate (Figure 1B). This was correlated with a block in differentiation (Figures 2A, B and D). This effect appeared to be mediated by the histone demethylase KDM4C (Figure 4D). The Reproducibility Project: Cancer Biology is a collaboration between the Center for Open Scienceand Science Exchange, and the results of the replications will be published by eLife.
DOI: http://dx.doi.org/10.7554/eLife.10860.001
doi:10.7554/eLife.10860
PMCID: PMC4805546  PMID: 26971564
IDH mutation; 2HG metabolism; reproducibility project: cancer biology; methodology; Human
23.  Genome-wide CRISPR screen in a mouse model of tumor growth and metastasis 
Cell  2015;160(6):1246-1260.
Summary
Genetic screens are powerful tools for identifying genes responsible for diverse phenotypes. Here we describe a genome-wide CRISPR-Cas9-mediated loss-of-function screen in tumor growth and metastasis. We mutagenized a non-metastatic mouse cancer cell line using a genome-scale library with 67,405 single guide RNAs (sgRNAs). The mutant cell pool rapidly generates metastases when transplanted into immunocompromised mice. Enriched sgRNAs in lung metastases and late stage primary tumors were found to target a small set of genes, suggesting specific loss-of-function mutations drive tumor growth and metastasis. Individual sgRNAs and a small pool of 624 sgRNAs targeting the top scoring genes from the primary screen dramatically accelerate metastasis. In all of these experiments, the effect of mutations on primary tumor growth positively correlates with the development of metastases. Our study demonstrates Cas9-based screening as a robust method to systematically assay gene phenotypes in cancer evolution in vivo.
doi:10.1016/j.cell.2015.02.038
PMCID: PMC4380877  PMID: 25748654
CRISPR/Cas9; metastasis; genome-wide screen; cancer evolution
24.  Regulation of Glutamine Carrier Proteins by RNF5 Determines Breast Cancer Response to ER Stress-inducing Chemotherapies 
Cancer cell  2015;27(3):354-369.
Summary
Many tumor cells are fueled by altered metabolism and increased glutamine (Gln) dependence. We identify regulation of the L-glutamine carrier proteins SLC1A5 and SLC38A2 (SLC1A5/38A2) by the ubiquitin ligase RNF5. Paclitaxel-induced ER stress to breast cancer (BCa) cells promotes RNF5 association, ubiquitination and degradation of SLC1A5/38A2. This decreases Gln uptake, levels of TCA cycle components, mTOR signaling and proliferation while increasing autophagy and cell death. Rnf5-deficient MMTV-PyMT mammary tumors were less differentiated and showed elevated SLC1A5 expression. Whereas RNF5 depletion in MDA-MB-231 cells promoted tumorigenesis and abolished paclitaxel responsiveness, SLC1A5/38A2 knockdown elicited opposing effects. Inverse RNF5HI/SLC1A5/38A2LO expression was associated with positive prognosis in BCa. Thus, RNF5 control of Gln uptake underlies BCa response to chemotherapies.
doi:10.1016/j.ccell.2015.02.006
PMCID: PMC4356903  PMID: 25759021
25.  Teardrop fracture following head-first impact in an ice hockey player: Case report and analysis of injury mechanisms 
Background
We report a case of a young male athlete who sustained a three column displaced teardrop fracture of the C5 vertebra due to a head-first impact in hockey, suffered neurapraxia, yet made full neurological recovery. This full recovery was in sharp contrast to multiple case series which reported permanent quadriplegia in the vast majority of teardrop fracture patients. We investigate the etiology and biomechanical mechanisms of injury.
Methods
Admission imaging revealed the teardrop fracture which consisted of: a frontal plane fracture which separated an anterior quadrilateral-shaped fragment from the posterior vertebral body; a vertical fracture of the posterior vertebral body in the sagittal plane; and incomplete fractures of the neural arch that initiated superiorly at the anterior aspect of the spinous process and left lamina adjacent to the superior facet. Epidural hematoma in the region of the C5 vertebra was observed in addition to disc and ligamentous disruptions at C4-5 and C5-6. Our patient was ultimately treated surgically with anterior fusion from C4 through C6 and subsequently with bilateral posterior fusion at C5-6.
Results
The injuries were caused by high-energy axial compression with the neck in a pre-flexed posture. The first fracture event consisted of the anterior vertebral body fragment being sheared off of the posterior fragment under the compression load due in part to the sagittal plane concavity of the C5 inferior endplate. The etiology of the vertical fracture of the posterior vertebral body fragment in the sagittal plane was consistent with a previously described hypothesis of the mechanistic injury events. First, the C4-5 disc height decreased under load which increased its hoop stress. Next, this increased hoop stress transferred lateral forces to the C5 uncinate processes which caused their outward expansion. Finally, the outward expansion of the uncinate processes caused the left and right sides of the vertebral body to split and spread. Evidence in support of this mechanistic event sequence was provided by the neural arch fractures which initiated superiorly, average angulation of the C5 uncinate processes, and similar well-established mechanisms causing vertical fractures at other spinal regions.
Conclusions
Our case study and analyses provide insight into the etiology of the specific teardrop fracture patterns observed clinically.
doi:10.14444/3009
PMCID: PMC4852594  PMID: 27162711
teardrop fracture; injury mechanism; Cervical Spine; hockey; impact biomechanics

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