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1.  Scratching the surface – tobacco-induced bacterial biofilms 
Tobacco Induced Diseases  2015;13(1):1.
Individual environmental factors, such as iron, temperature and oxygen, are known to have a profound effect on bacterial phenotype. Therefore, it is surprising so little known is about the influence of chemically complex cigarette smoke on bacterial physiology. Recent evidence has demonstrated that tobacco smoke and components alter the bacterial surface and promote biofilm formation in several important human pathogens, including Staphylococcus aureus, Streptococcus mutans, Klebsiella pneumonia, Porphyromonas gingivalis and Pseudomonas aeruginosa. The mechanisms underlying this phenomenon and the relevance to increased susceptibility to infectious disease in smokers and to treatment are reviewed.
doi:10.1186/s12971-014-0026-3
PMCID: PMC4323140  PMID: 25670926
Bacteria; Biofilms; Cigarette smoking; Infectious diseases; Outer membrane; Tobacco
2.  Comprehensive miRNA sequence analysis reveals survival differences in diffuse large B-cell lymphoma patients 
Genome Biology  2015;16(1):18.
Background
Diffuse large B-cell lymphoma (DLBCL) is an aggressive disease, with 30% to 40% of patients failing to be cured with available primary therapy. microRNAs (miRNAs) are RNA molecules that attenuate expression of their mRNA targets. To characterize the DLBCL miRNome, we sequenced miRNAs from 92 DLBCL and 15 benign centroblast fresh frozen samples and from 140 DLBCL formalin-fixed, paraffin-embedded tissue samples for validation.
Results
We identify known and candidate novel miRNAs, 25 of which are associated with survival independently of cell-of-origin and International Prognostic Index scores, which are established indicators of outcome. Of these 25 miRNAs, six miRNAs are significantly associated with survival in our validation cohort. Abundant expression of miR-28-5p, miR-214-5p, miR-339-3p, and miR-5586-5p is associated with superior outcome, while abundant expression of miR-324-5p and NOVELM00203M is associated with inferior outcome. Comparison of DLBCL miRNA-seq expression profiles with those from other cancer types identifies miRNAs that were more abundant in B-cell contexts. Unsupervised clustering of miRNAs identifies two clusters of patients that have distinct differences in their outcomes. Our integrative miRNA and mRNA expression analyses reveal that miRNAs increased in abundance in DLBCL appear to regulate the expression of genes involved in metabolism, cell cycle, and protein modification. Additionally, these miRNAs, including one candidate novel miRNA, miR-10393-3p, appear to target chromatin modification genes that are frequent targets of somatic mutation in non-Hodgkin lymphomas.
Conclusions
Our comprehensive sequence analysis of the DLBCL miRNome identifies candidate novel miRNAs and miRNAs associated with survival, reinforces results from previous mutational analyses, and reveals regulatory networks of significance for lymphomagenesis.
Electronic supplementary material
The online version of this article (doi:10.1186/s13059-014-0568-y) contains supplementary material, which is available to authorized users.
doi:10.1186/s13059-014-0568-y
PMCID: PMC4308918
3.  Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells 
Nature biotechnology  2014;32(7):670-676.
Bacterial type II CRISPR-Cas9 systems have been widely adapted for RNA- guided genome editing and transcription regulation in eukaryotic cells, yet their in vivo target specificity is poorly understood. Here we mapped genome-wide binding sites of a catalytically inactive Cas9 (dCas9) from Streptococcus pyogenes loaded with single guide RNAs (sgRNAs) in mouse embryonic stem cells (mESCs). Each of the four sgRNAs tested targets dCas9 to tens to thousands of genomic sites, characterized by a 5-nucleotide seed region in the sgRNA, in addition to an NGG protospacer adjacent motif (PAM). Chromatin inaccessibility prevents dCas9 binding to other sites with matching seed sequences, and consequently 70% of off-target sites are associated with genes. Targeted sequencing of 295 dCas9 binding sites in mESCs transfected with catalytically active Cas9 identified only one site mutated above background. We propose a two-state model for Cas9 binding and cleavage, in which a seed match triggers binding but extensive pairing with target DNA is required for cleavage.
doi:10.1038/nbt.2889
PMCID: PMC4145672  PMID: 24752079
4.  Accumulation of Oxidized LDL in the Tendon Tissues of C57BL/6 or Apolipoprotein E Knock-Out Mice That Consume a High Fat Diet: Potential Impact on Tendon Health 
PLoS ONE  2014;9(12):e114214.
Objective
Clinical studies have suggested an association between dyslipidemia and tendon injuries or chronic tendon pain; the mechanisms underlying this association are not yet known. The objectives of this study were (1) to evaluate the impact of a high fat diet on the function of load-bearing tendons and on the distribution in tendons of oxidized low density lipoprotein (oxLDL), and (2) to examine the effect of oxLDL on tendon fibroblast proliferation and gene expression.
Methods
Gene expression (Mmp2, Tgfb1, Col1a1, Col3a1), fat content (Oil Red O staining), oxLDL levels (immunohistochemistry) and tendon biomechanical properties were examined in mice (C57Bl/6 or ApoE -/-) receiving a standard or a high fat diet. Human tendon fibroblast proliferation and gene expression (COL1A1, COL3A1, MMP2) were examined following oxLDL exposure.
Results
In both types of mice (C57Bl/6 or ApoE -/-), consumption of a high fat diet led to a marked increase in oxLDL deposition in the load-bearing extracellular matrix of the tendon. The consumption of a high fat diet also reduced the failure stress and load of the patellar tendon in both mouse types, and increased Mmp2 expression. ApoE -/- mice exhibited more pronounced reductions in tendon function than wild-type mice, and decreased expression of Col1a1 compared to wild type mice. Human tendon fibroblasts responded to oxLDL by increasing their proliferation and their mRNA levels of MMP2, while decreasing their mRNA levels for COL1A1 and COL3A1.
Conclusion
The consumption of a high fat diet resulted in deleterious changes in tendon function, and these changes may be explained in part by the effects of oxLDL, which induced a proliferative, matrix-degrading phenotype in human tenocytes.
doi:10.1371/journal.pone.0114214
PMCID: PMC4264764  PMID: 25502628
5.  The Surgical Treatment of Single Level Multi-Focal Subarticular and Paracentral and/or Far-Lateral Lumbar Disc Herniations: The Single Incision Full Endoscopic Approach 
Background
Surgery for same level multi-focal extruded lumbar disc herniations is technically challenging and the optimal method controversial. The subarticular disc herniation may pose the most challenging subtype requiring partial or complete facetectomy with or without fusion. The far-lateral disc herniation, often treated using a Wiltse approach, can also be difficult to access especially in the obese patient. When both the subarticular and far-lateral subtypes are simultaneously present at the same level with or without a paracentral disc herniation, a total facetectomy and interbody fusion (TLIF) or a total disc replacement (TDR) may be necessary. Endoscopic surgical techniques may reduce the need for these more invasive methods.
Methods
Fifteen patients (6 male and 9 female) who had same level multi-focal (subarticular as well as far-lateral and/or paracentral) extruded disc herniations underwent single incision unilateral endoscopic disc excision by the same surgeon at a single institution. Patients were prospectively followed for an average of 15.3 months (range 14-18 months) and outcomes were evaluated radiographically and clinically (Visual Analogue Scale (VAS) and Oswestry Disability Index (ODI).
Results
The mean operative time was 52 minutes with minimal blood loss in all cases. Fourteen of the 15 patients were discharged to home on the day of their surgery. The mean ODI and leg VAS scores improved from 22.9 ± 3.2 to 12.9 ± 2.7 (p < 0.005), and from 8.6 ± 1.6 to 2.1 + 0.4 (p < 0.005), respectively.
Conclusions
After an average of 15.3 months of follow-up, the clinical and radiographic results of full endoscopic surgical treatment of single level multi-focal (subarticular as well as far-lateral and/or paracentral) disc herniations are excellent. This study is a case series with mid-term follow-up (Level IV).
Clinical Relevance
Foraminal and extra-foraminal full endoscopic decompression appears to offer a safe minimally invasive solution to a complex pathologic problem.
doi:10.14444/1016
PMCID: PMC4325509
Endoscopic Spine Surgery; Lumbar; Far-lateral; Subarticular; Paracentral; Disc Herniation
6.  The effectiveness and cost-effectiveness of treatments for idiopathic pulmonary fibrosis: systematic review, network meta-analysis and health economic evaluation 
Background
Idiopathic pulmonary fibrosis (IPF) is a life-limiting lung disease with considerable impact on patients and carers as the disease progresses. Currently few treatments are available. We aimed to evaluate the clinical and cost-effectiveness of available treatments for IPF.
Methods
Systematic reviews of clinical effectiveness, quality of life and cost effectiveness were undertaken. Eleven bibliographic databases were searched from inception to July 2013 and studies were assessed for eligibility against a set of pre-defined criteria. Two reviewers screened references, extracted data from included studies and appraised their quality. An advisory group was consulted about the choice of interventions. A narrative review was undertaken and where feasible fixed effect and random effects meta-analysis were undertaken including a network meta-analysis (NMA).
A decision-analytic Markov model was developed to estimate cost-effectiveness of pharmacological treatments for IPF. Following best practice recommendations, the model perspective was of the national health service and personal social services, a discount rate of 3.5% for costs and health benefits was applied and outcomes were expressed as cost per quality adjusted life-year gained. Parameter values were obtained from the NMA and systematic reviews. Sensitivity analyses were undertaken.
Results
Fourteen studies were included in the review of clinical effectiveness, of which one evaluated azathioprine, three N-acetylcysteine [NAC] (alone or in combination), four pirfenidone, one nintedanib, one sildenafil, one thalidomide, two pulmonary rehabilitation, and one a disease management programme. Study quality was generally good. Evidence suggests that some effective treatments are available. In NMA only nintedanib and pirfenidone show statistically significant improvements. The model results show increased survival for five pharmacological treatments (NAC triple therapy, inhaled NAC, nintedanib, pirfenidone, and sildenafil) compared with best supportive care, at increased cost. Only inhaled NAC was cost-effective at current willingness to pay thresholds but it may not be clinically effective.
Conclusions
Few interventions have any statistically significant effect and the cost-effectiveness of treatments is uncertain. A lack of studies on palliative care approaches was identified and there is a need for further research into pulmonary rehabilitation and thalidomide in particular. A well conducted RCT on inhaled NAC therapy should also be considered.
Electronic supplementary material
The online version of this article (doi:10.1186/2050-6511-15-63) contains supplementary material, which is available to authorized users.
doi:10.1186/2050-6511-15-63
PMCID: PMC4247619  PMID: 25410822
Idiopathic pulmonary fibrosis; Systematic review; Meta-analysis; Cost-effectiveness
7.  Engineering a reagentless biosensor for single-stranded DNA to measure real-time helicase activity in Bacillus 
Biosensors & Bioelectronics  2014;61(100):579-586.
Single-stranded DNA-binding protein (SSB) is a well characterized ubiquitous and essential bacterial protein involved in almost all aspects of DNA metabolism. Using the Bacillus subtilis SSB we have generated a reagentless SSB biosensor that can be used as a helicase probe in B. subtilis and closely related gram positive bacteria. We have demonstrated the utility of the probe in a DNA unwinding reaction using a helicase from Bacillus and for the first time, characterized the B. subtilis SSB's DNA binding mode switching and stoichiometry. The importance of SSB in DNA metabolism is not limited to simply binding and protecting ssDNA during DNA replication, as previously thought. It interacts with an array of partner proteins to coordinate many different aspects of DNA metabolism. In most cases its interactions with partner proteins is species-specific and for this reason, knowing how to produce and use cognate reagentless SSB biosensors in different bacteria is critical. Here we explain how to produce a B. subtilis SSB probe that exhibits 9-fold fluorescence increase upon binding to single stranded DNA and can be used in all related gram positive firmicutes which employ drastically different DNA replication and repair systems than the widely studied Escherichia coli. The materials to produce the B. subtilis SSB probe are commercially available, so the methodology described here is widely available unlike previously published methods for the E. coli SSB.
Highlights
•Bacillus subtilis specific biosensor has been produced.•Problem posed by an internal cysteine residue was overcome by site-specific mutagenesis.•FDA5M is commercially available enhancing the production of B. subtilis biosensor..•B. subtilis SSB exhibits salt dependent DNA-binding mode switch like E. coli SSB.•The B. subtilis SSB biosensor can be used in cognate reactions and related firmicutes.
doi:10.1016/j.bios.2014.06.011
PMCID: PMC4103019  PMID: 24953846
DNA, (deoxynucleic acid); SSB, (single strand DNA binding protein); ssDNA, (single stranded DNA); IDCC, N-[2-(iodoacetamido)ethyl]-7-diethylaminocoumarin3-carboxamide; MDCC, 7-diethylamino-3-((((2-maleimidyl)ethyl)amino)carbonyl) coumarin; FDA5M, fluorescein diacetate 5 maleimide; EMSA, electrophoretic mobility shift assay; DTT, dithiothreitol; EDTA, ethelenediaminetetraacetic acid; TBE, Tris boric acid ethelenediaminetetraacetic acid; SSB; Helicases; Fluorescence; DNA unwinding; Bacillus subtilis; Firmicutes
8.  Robust Fitting of a Weibull Model with Optional Censoring 
The Weibull family is widely used to model failure data, or lifetime data, although the classical two-parameter Weibull distribution is limited to positive data and monotone failure rate. The parameters of the Weibull model are commonly obtained by maximum likelihood estimation; however, it is well-known that this estimator is not robust when dealing with contaminated data. A new robust procedure is introduced to fit a Weibull model by using L2 distance, i.e. integrated square distance, of the Weibull probability density function. The Weibull model is augmented with a weight parameter to robustly deal with contaminated data. Results comparing a maximum likelihood estimator with an L2 estimator are given in this article, based on both simulated and real data sets. It is shown that this new L2 parametric estimation method is more robust and does a better job than maximum likelihood in the newly proposed Weibull model when data are contaminated. The same preference for L2 distance criterion and the new Weibull model also happens for right-censored data with contamination.
doi:10.1016/j.csda.2013.05.009
PMCID: PMC3718081  PMID: 23888090
Weibull distribution; L2 distance; Robust estimator; Maximum likelihood; Right-censored data; Contamination
9.  The role of ExbD in periplasmic pH homeostasis in Helicobacter pylori 
Helicobacter  2013;18(5):363-372.
Background
Helicobacter pylori, a neutralophile, colonizes the acidic environment of the human stomach by employing acid acclimation mechanisms that regulate periplasmic and cytoplasmic pH. The regulation of urease activity is central to acid acclimation. Inactive urease apoenzyme, UreA/B, requires nickel for activation. Accessory proteins UreE, F, G and H are required for nickel insertion into apoenzyme. The ExbB/ExbD/TonB complex transfers energy from the inner to outer membrane, providing the driving force for nickel uptake. Therefore, the aim of this study was to determine the contribution of ExbD to pH homeostasis.
Materials and Methods
A nonpolar exbD knockout was constructed and survival, growth, urease activity, and membrane potential were determined in comparison to wildtype.
Results
Survival of the ΔexbD strain was significantly reduced at pH 3.0. Urease activity as a function of pH and UreI activation were similar to the wildtype strain, showing normal function of the proton-gated urea channel, UreI. The increase in total urease activity over time in acid seen in the wildtype strain was abolished in the ΔexbD strain, but recovered in the presence of supra-physiologic nickel concentrations, demonstrating that the effect of the ΔexbD mutant is due to loss of a necessary constant supply of nickel. In acid, ΔexbD also decreased its ability to maintain membrane potential and periplasmic buffering in the presence of urea.
Conclusions
ExbD is essential for maintenance of periplasmic buffering and membrane potential by transferring energy required for nickel uptake, making it a potential non-antibiotic target for H. pylori eradication.
doi:10.1111/hel.12055
PMCID: PMC3742559  PMID: 23600974
10.  Health Assessment Questionnaire disability progression in early rheumatoid arthritis: Systematic review and analysis of two inception cohorts 
Objective
The Health Assessment Questionnaire is widely used for patients with inflammatory polyarthritis (IP) and its subset, rheumatoid arthritis (RA). In this study, we evaluated the progression of HAQ scores in RA (i) by systematically reviewing the published literature on the methods used to assess changes in functional disability over time and (ii) to study in detail HAQ progression in two large prospective observational studies from the UK.
Methods
Data from two large inception cohorts, ERAS and NOAR, were studied to determine trajectories of HAQ progression over time by applying latent class growth models (LCGMs) to each dataset separately. Age, sex, baseline DAS28, symptom duration, rheumatoid factor, fulfilment of the 1987 ACR criteria and socio-economic status (SES) were included as potential predictors of HAQ trajectory subgroup membership.
Results
The literature search identified 49 studies showing that HAQ progression has mainly been based on average changes in the total study population. In the HAQ progression study, a LCGM with four HAQ trajectory subgroups was selected as providing the best fit in both cohorts. In both the cohorts, older age, female sex, longer symptom duration, fulfilment of the 1987 ACR criteria, higher DAS28 and lower SES were associated with increased likelihood of membership of subgroups with worse HAQ progression.
Conclusion
Four distinct HAQ trajectory subgroups were derived from the ERAS and NOAR cohorts. The fact that the subgroups identified were nearly identical supports their validity. Identifying distinct groups of patients who are at risk of poor functional outcome may help to target therapy to those who are most likely to benefit.
doi:10.1016/j.semarthrit.2014.05.003
PMCID: PMC4282305  PMID: 24925692
Systematic review; Latent class growth model; Health Assessment Questionnaire; Rheumatoid arthritis; Inflammatory polyarthritis; Predictors
11.  Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity 
Cell  2013;154(6):10.1016/j.cell.2013.08.021.
Targeted genome editing technologies have enabled a broad range of research and medical applications. The Cas9 nuclease from the microbial CRISPR-Cas system is targeted to specific genomic loci by a 20-nt guide sequence, which can tolerate certain mismatches to the DNA target and thereby promote undesired off-target mutagenesis. Here, we describe an approach that combines a Cas9 nickase mutant with pairs of guide RNAs to introduce targeted double-strand breaks. Given that individual nicks in the genome are repaired with high fidelity, simultaneous nicking via appropriately offset guide RNAs effectively extends the number of specifically recognized bases in the target site. We demonstrate that paired nicking can be used to reduce off-target activity by 50–1,000 fold in cell lines and facilitate gene knockout in mouse zygotes without sacrificing on-target cleavage efficiency. This versatile strategy thus enables a wide variety of genome editing applications with higher levels of specificity.
doi:10.1016/j.cell.2013.08.021
PMCID: PMC3856256  PMID: 23992846
12.  Mechanism-Based Epigenetic Chemosensitization Therapy of Diffuse Large B Cell Lymphoma 
Cancer discovery  2013;3(9):1002-1019.
Although aberrant DNA methylation patterning is a hallmark of cancer, the relevance of targeting DNA methyltransferases (DNMT) remains unclear for most tumors. In diffuse large B-cell lymphoma (DLBCL) we observed that chemo-resistance is associated with aberrant DNA methylation programming. Prolonged exposure to low-dose DNMT inhibitors (DNMTIs) reprogrammed chemo-resistant cells to become doxorubicin sensitive without major toxicity in vivo. Nine genes were recurrently hypermethylated in chemo-resistant DLBCL. Of these, SMAD1 was a critical contributor, and reactivation was required for chemosensitization. A phase I clinical study was performed evaluating azacitidine priming followed by standard chemoimmunotherapy in high-risk newly diagnosed DLBCL patients. The combination was well tolerated and yielded a high rate of complete remission. Pre and post azacitidine treatment biopsies confirmed SMAD1 demethylation and chemosensitization, delineating a personalized strategy for the clinical use of DNMTIs.
doi:10.1158/2159-8290.CD-13-0117
PMCID: PMC3770813  PMID: 23955273
Non-Hodgkin lymphoma; epigenetic; DNA methylation; DNMT inhibitor; chemoresistance
13.  STAT6 controls the number of regulatory T cells in vivo thereby regulating allergic lung inflammation1 
STAT6 plays a central role in IL-4-mediated allergic responses. Several studies indicate that regulatory T cells (Treg) can be modulated by IL-4 in vitro. We previously showed that STAT6−/− mice are highly resistant to allergic lung inflammation even when wild type Th2 effectors were provided and that they have increased numbers of Tregs. However, the role of STAT6 in modulating Tregs in vivo during allergic lung inflammation has not been thoroughly investigated. To investigate Treg and STAT6 interaction during allergic inflammation, STAT6−/−, STAT6×RAG2−/− and RAG2−/− mice were subjected to OVA sensitization and challenge following adoptive transfer of OVA-specific, wild type Th2 effectors with or without prior Treg depletion/ inactivation using anti-CD25 (PC61). As expected, STAT6−/− mice were highly resistant to airway inflammation and remodeling. In contrast, allergic lung inflammation was partially restored in STAT6−/− mice treated with PC61 to levels observed in STAT6×RAG2−/− mice. In some cases, STAT6×RAG2−/− mice were also given natural (n) Tregs along with Th2 effectors. Adoptive transfer of nTregs caused a substantial reduction in BAL eosinophil composition and suppressed airway remodeling and T cell migration into the lung in STAT6×RAG2−/− mice to levels comparable to those in STAT6−/− mice. These results demonstrate the STAT6-dependent suppression of Tregs in vivo in order to promote allergic airway inflammation.
doi:10.4049/jimmunol.1300486
PMCID: PMC3735779  PMID: 23825312
14.  Activity-induced convergence of APP and BACE-1 in acidic microdomains via an endocytosis-dependent pathway 
Neuron  2013;79(3):447-460.
The convergence of APP (substrate) and BACE-1 (enzyme) is a rate-limiting, obligatory event triggering the amyloidogenic pathway – a key step in Alzheimer’s disease (AD) pathology. However, as both APP/BACE-1 are highly expressed in brain, mechanisms precluding their unabated convergence are unclear. Exploring dynamic localization of APP/BACE-1 in cultured hippocampal neurons, we found that after synthesis via the secretory-pathway, dendritic APP/BACE-1-containing vesicles are largely segregated in physiologic states. While BACE-1 is largely sorted into acidic recycling endosomes, APP is conveyed in Golgi-derived vesicles. However upon activity-induction – a known trigger of the amyloidogenic pathway – APP is routed into BACE-1-positive recycling endosomes via a clathrin-dependent mechanism. A partitioning/convergence of APP/BACE-1 vesicles is also apparent in control/AD brains respectively. Considering BACE-1 is optimally active in an acidic environment, our experiments suggest that neurons have evolved trafficking strategies that normally limit APP/BACE-1 proximity; and also uncover a pathway routing APP into BACE-1-containing organelles – triggering amyloidogenesis.
doi:10.1016/j.neuron.2013.05.035
PMCID: PMC3741682  PMID: 23931995
amyloid precursor protein (APP); β-site APP cleaving enzyme (BACE-1); vesicle trafficking; neuronal activity; recycling endosomes
15.  The Role of Janus Kinase (JAK)-3 in Regulating Toll-Like Receptor-Mediated Inflammatory Cytokine Production in Innate Immune Cells 
The role of Janus kinase (JAK)-3 in TLR-mediated innate immune responses is poorly understood, although the suppressive function of JAK3 inhibition in adaptive immune response has been well studied. In this study, we found that JAK3 inhibition enhanced TLR-mediated immune responses by differentially regulating pro- and anti- inflammatory cytokine production in innate immune cells. Specifically, JAK3 inhibition by pharmacological inhibitors or specific siRNA, or JAK3 gene knockout resulted in an increase in TLR-mediated production of pro-inflammatory cytokines, while concurrently decreasing the production of IL-10. Inhibition of JAK3 suppressed phosphorylation of PI3 kinase downstream effectors including Akt, mTORC1, GSK3β and CREB. Constitutive activation of Akt or inhibition of GSK3β abrogated the capability of JAK3 inhibition to enhance pro-inflammatory cytokines and suppress IL-10 production. In contrast, inhibition of PI3K enhanced this regulatory ability of JAK3 in LPS stimulated monocytes. At the transcriptional level, JAK3 knockout lead to the increased phosphorylation of STATs that could be attenuated by neutralization of de novo inflammatory cytokines. JAK3 inhibition exhibited a GSK3 activity-dependent ability to enhance phosphorylation levels and DNA binding of NF-κB p65. Moreover, JAK3 inhibition correlated with an increased CD4+ T cell response. Additionally, higher neutrophil infiltration, IL-17 expression, and intestinal epithelium erosion were observed in JAK3 knockout mice. These findings demonstrate the negative regulatory function of JAK3, and elucidate the signaling pathway by which JAK3 differentially regulates TLR-mediated inflammatory cytokine production in innate immune cells.
doi:10.4049/jimmunol.1203084
PMCID: PMC3720823  PMID: 23797672
JAK3; PI3K; GSK3; TLR4; Inflammatory cytokines
16.  N-terminal Domain of Prion Protein Directs Its Oligomeric Association* 
The Journal of Biological Chemistry  2014;289(37):25497-25508.
Background: Non-fibrillar oligomers are implicated as neurotoxic species in several amyloid neurodegenerative diseases.
Results: Full-length prion protein (PrP) forms distinct non-fibrillar β-sheet-rich oligomers. Truncated protein, lacking the N terminus forms nonspecific aggregates.
Conclusion: The unstructured N terminus of PrP is key to the folding and aggregation of its structured region.
Significance: To examine the full repertoire of PrP conformers and assembly states, full-length protein should be used.
The self-association of prion protein (PrP) is a critical step in the pathology of prion diseases. It is increasingly recognized that small non-fibrillar β-sheet-rich oligomers of PrP may be of crucial importance in the prion disease process. Here, we characterize the structure of a well defined β-sheet-rich oligomer, containing ∼12 PrP molecules, and often enclosing a central cavity, formed using full-length recombinant PrP. The N-terminal region of prion protein (residues 23–90) is required for the formation of this distinct oligomer; a truncated form comprising residues 91–231 forms a broad distribution of aggregated species. No infectivity or toxicity was found using cell and animal model systems. This study demonstrates that examination of the full repertoire of conformers and assembly states that can be accessed by PrP under specific experimental conditions should ideally be done using the full-length protein.
doi:10.1074/jbc.M114.566588
PMCID: PMC4162156  PMID: 25074940
Amyloid; Intrinsically Disordered Protein; Prion; Protein Aggregation; Protein Folding; β-PrP; β-Sheet; CJD; Molten Globule; Oligomer
17.  Microdialysis Sampling Coupled to Microchip Electrophoresis with Integrated Amperometric Detection on an All Glass Substrate 
The development of an all-glass separation-based sensor using microdialysis coupled to microchip electrophoresis with amperometric detection is described. The system includes a flow-gated interface to inject discrete sample plugs from the microdialysis perfusate into the microchip electrophoresis system. Electrochemical detection was accomplished with a platinum electrode in an in-channel configuration using a wireless electrically isolated potentiostat. To facilitate bonding around the in-channel electrode, a fabrication process was employed that produced a working and a reference electrode flush with the glass surface. Both normal and reversed polarity separations were performed with this sensor. The system was evaluated in vitro for the continuous monitoring of the production of hydrogen peroxide from the reaction of glucose oxidase with glucose. Microdialysis experiments were performed using a BASi loop probe with an overall lag time of approximately five minutes and a rise time of less than 60 seconds.
doi:10.1002/cphc.201300449
PMCID: PMC4000424  PMID: 23794474
glass microchip; electrochemical detection; microchip electrophoresis; microdialysis; microfluidics
18.  Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells 
Science (New York, N.Y.)  2013;343(6166):84-87.
The simplicity of programming the CRISPR-associated nuclease Cas9 to modify specific genomic loci suggests a new way to interrogate gene function on a genome-wide scale. We show that lentiviral delivery of a genome-scale CRISPR-Cas9 knockout (GeCKO) library targeting 18,080 genes with 64,751 unique guide sequences enables both negative and positive selection screening in human cells. First, we used the GeCKO library to identify genes essential for cell viability in cancer and pluripotent stem cells. Next, in a melanoma model, we screened for genes whose loss is involved in resistance to vemurafenib, a therapeutic that inhibits mutant protein kinase BRAF. Our highest-ranking candidates include previously validated genes NF1 and MED12 as well as novel hits NF2, CUL3, TADA2B, and TADA1. We observe a high level of consistency between independent guide RNAs targeting the same gene and a high rate of hit confirmation, demonstrating the promise of genome-scale screening with Cas9.
doi:10.1126/science.1247005
PMCID: PMC4089965  PMID: 24336571
21.  Identifying solutions to increase participation in physical activity interventions within a socio-economically disadvantaged community: a qualitative study 
Background
There is an urgent need to increase population levels of physical activity, particularly amongst those who are socio-economically disadvantaged. Multiple factors influence physical activity behaviour but the generalisability of current evidence to such ‘hard-to-reach’ population subgroups is limited by difficulties in recruiting them into studies. Also, rigorous qualitative studies of lay perceptions and perceptions of community leaders about public health efforts to increase physical activity are sparse. We sought to explore, within a socio-economically disadvantaged community, residents’ and community leaders’ perceptions of physical activity (PA) interventions and issues regarding their implementation, in order to improve understanding of needs, expectations, and social/environmental factors relevant to future interventions.
Methods
Within an ongoing regeneration project (Connswater Community Greenway), in a socio-economically disadvantaged community in Belfast, we collaborated with a Community Development Agency to purposively sample leaders from public- and voluntary-sector community groups and residents. Individual semi-structured interviews were conducted with 12 leaders. Residents (n = 113), of both genders and a range of ages (14 to 86 years) participated in focus groups (n = 14) in local facilities. Interviews and focus groups were recorded, transcribed verbatim and analysed using a thematic framework.
Results
Three main themes were identified: awareness of PA interventions; factors contributing to intervention effectiveness; and barriers to participation in PA interventions. Participants reported awareness only of interventions in which they were involved directly, highlighting a need for better communications, both inter- and intra-sectoral, and with residents. Meaningful engagement of residents in planning/organisation, tailoring to local context, supporting volunteers, providing relevant resources and an ‘exit strategy’ were perceived as important factors related to intervention effectiveness. Negative attitudes such as apathy, disappointing experiences, information with no perceived personal relevance and limited access to facilities were barriers to people participating in interventions.
Conclusions
These findings illustrate the complexity of influences on a community’s participation in PA interventions and support a social-ecological approach to promoting PA. They highlight the need for cross-sector working, effective information exchange, involving residents in bottom-up planning and providing adequate financial and social support. An in-depth understanding of a target population’s perspectives is of key importance in translating PA behaviour change theories into practice.
doi:10.1186/1479-5868-11-68
PMCID: PMC4038056  PMID: 24886604
Physical activity; Interventions; Socio-economic disadvantage; Community; Public health; Qualitative; Health information; Communication; Cross-sector working
22.  Structure and Catalytic Regulatory Function of Ubiquitin Specific Protease 11 N-Terminal and Ubiquitin-like Domains 
Biochemistry  2014;53(18):2966-2978.
The ubiquitin specific protease 11 (USP11) is implicated in DNA repair, viral RNA replication, and TGFβ signaling. We report the first characterization of the USP11 domain architecture and its role in regulating the enzymatic activity. USP11 consists of an N-terminal “domain present in USPs” (DUSP) and “ubiquitin-like” (UBL) domain, together referred to as DU domains, and the catalytic domain harboring a second UBL domain. Crystal structures of the DU domains show a tandem arrangement with a shortened β-hairpin at the two-domain interface and altered surface characteristics compared to the homologues USP4 and USP15. A conserved VEVY motif is a signature feature at the two-domain interface that shapes a potential protein interaction site. Small angle X-ray scattering and gel filtration experiments are consistent with the USP11DU domains and full-length USP11 being monomeric. Unexpectedly, we reveal, through kinetic assays of a series of deletion mutants, that the catalytic activity of USP11 is not regulated through intramolecular autoinhibition or activation by the N-terminal DU or UBL domains. Moreover, ubiquitin chain cleavage assays with all eight linkages reveal a preference for Lys63-, Lys6-, Lys33-, and Lys11-linked chains over Lys27-, Lys29-, and Lys48-linked and linear chains consistent with USP11’s function in DNA repair pathways that is mediated by the protease domain. Our data support a model whereby USP11 domains outside the catalytic core domain serve as protein interaction or trafficking modules rather than a direct regulatory function of the proteolytic activity. This highlights the diversity of USPs in substrate recognition and regulation of ubiquitin deconjugation.
doi:10.1021/bi500116x
PMCID: PMC4020902  PMID: 24724799
23.  EZH2 is required for germinal center formation and somatic EZH2 mutations promote lymphoid transformation 
Cancer cell  2013;23(5):677-692.
The EZH2 histone methyltransferase is highly expressed in germinal center (GC) B-cells and targeted by somatic mutations in B-cell lymphomas. Here we find that EZH2 deletion or pharmacologic inhibition suppresses GC formation and functions in mice. EZH2 represses proliferation checkpoint genes and helps establish bivalent chromatin domains at key regulatory loci to transiently suppress GC B-cell differentiation. Somatic mutations reinforce these physiological effects through enhanced silencing of EZH2 targets in B-cells, and in human B-cell lymphomas. Conditional expression of mutant EZH2 in mice induces GC hyperplasia and accelerated lymphomagenesis in cooperation with BCL2. GCB-type DLBCLs are mostly addicted to EZH2, regardless of mutation status, but not the more differentiated ABC-type DLBCLs, thus clarifying the therapeutic scope of EZH2 targeting.
doi:10.1016/j.ccr.2013.04.011
PMCID: PMC3681809  PMID: 23680150
24.  Anomalous Small Angle X-Ray Scattering Simulations: Proof of Concept for Distance Measurements for Nanoparticle-Labelled Biomacromolecules in Solution 
PLoS ONE  2014;9(4):e95664.
Anomalous small angle X-ray scattering can in principle be used to determine distances between metal label species on biological molecules. Previous experimental studies in the past were unable to distinguish the label-label scattering contribution from that of the molecule, because of the use of atomic labels; these labels contribute only a small proportion of the total scattering signal. However, with the development of nanocrystal labels (of 50–100 atoms) there is the possibility for a renewed attempt at applying anomalous small angle X-ray scattering for distance measurement. This is because the contribution to the scattered signal is necessarily considerably stronger than for atomic labels. Here we demonstrate through simulations, the feasibility of the technique to determine the end-to-end distances of labelled nucleic acid molecules as well as other internal distances mimicking a labelled DNA binding protein if the labels are dissimilar metal nanocrystals. Of crucial importance is the ratio of mass of the nanocrystals to that of the labelled macromolecule, as well as the level of statistical errors in the scattering intensity measurements. The mathematics behind the distance determination process is presented, along with a fitting routine than incorporates maximum entropy regularisation.
doi:10.1371/journal.pone.0095664
PMCID: PMC3997412  PMID: 24759797
25.  Neutrophils alter epithelial response to Porphyromonas gingivalis in a gingival crevice model 
Molecular oral microbiology  2012;28(2):102-113.
Summary
A gingival crevice model (epithelial cell- Porphyromonas gingivalis – neutrophil) was established and used to profile gingipain, matrix metalloproteinase, MMP mediators (NGAL and TIMP-1) and cytokine networks. Smoking is the primary environmental risk factor for periodontitis. Therefore, the influence of cigarette smoke extract (CSE) was also monitored in the same model. P. gingivalis alone induced low levels of IL-1β and IL-8 from epithelial cells, but high levels of both cytokines were produced on the addition of neutrophils. CSE-exposure (100 and 1000 ng/ml nicotine equivalency) significantly compromised P. gingivalis-induced cytokine secretion (both p < 0.05). P. gingivalis induced impressive secretion of NGAL (p < 0.05) which was not influenced by CSE. The influence of CSE on gingipains production was strain-specific. Purified gingipains effectively and rapidly degraded both TIMP-1 and MMP-9. Induction of large amounts of NGAL, degradation of TIMP-1, and increased gingipain activity would each be expected to prolong collagen degradation and promote disease progression. However, gingipains also degrade MMP-9. Thus, P. gingivalis exerts a complex influence on the proteolytic balance of a gingival crevice model. CSE-exposure reduces the pro-inflammatory cytokine burden, which may be expected to promote P. gingivalis survival. In addition to novel findings that provide mechanistic insight into periodontal disease progression, these results are in keeping with the recognized clinical dogma of decreased inflammation / increased disease in smokers. Thus, this straightforward gingival crevice model is established as a suitable vehicle for the elucidation of mechanisms that contribute to susceptibility to periodontitis.
doi:10.1111/omi.12008
PMCID: PMC3594541  PMID: 23193955
cytokines; epithelial cells; neutrophils; matrix metalloproteinases; periodontitis; P. gingivalis; tobacco smoke

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