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1.  Neutrophils alter epithelial response to Porphyromonas gingivalis in a gingival crevice model 
Molecular oral microbiology  2012;28(2):102-113.
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.
PMCID: PMC3594541  PMID: 23193955
cytokines; epithelial cells; neutrophils; matrix metalloproteinases; periodontitis; P. gingivalis; tobacco smoke
2.  DNA targeting specificity of RNA-guided Cas9 nucleases 
Nature biotechnology  2013;31(9):827-832.
The Streptococcus pyogenes Cas9 (SpCas9) nuclease can be efficiently targeted to genomic loci by means of singleguide RNAs (sgRNAs) to enable genome editing1–10. Here, we characterize SpCas9 targeting specificity in human cells to inform the selection of target sites and avoid off-target effects. Our study evaluates >700 guide RNA variants and SpCas9-induced indel mutation levels at >100 predicted genomic off-target loci in 293T and 293FT cells. We find that SpCas9 tolerates mismatches between guide RNA and target DNA at different positions in a sequence-dependent manner, sensitive to the number, position and distribution of mismatches. We also show that SpCas9-mediated cleavage is unaffected by DNA methylation and that the dosage of SpCas9 and sgRNA can be titrated to minimize off-target modification. To facilitate mammalian genome engineering applications, we provide a web-based software tool to guide the selection and validation of target sequences as well as off-target analyses.
PMCID: PMC3969858  PMID: 23873081
3.  Genome engineering using the CRISPR-Cas9 system 
Nature protocols  2013;8(11):2281-2308.
Targeted nucleases are powerful tools for mediating genome alteration with high precision. The RNA-guided Cas9 nuclease from the microbial clustered regularly interspaced short palindromic repeats (CRISPR) adaptive immune system can be used to facilitate efficient genome engineering in eukaryotic cells by simply specifying a 20-nt targeting sequence within its guide RNA. Here we describe a set of tools for Cas9-mediated genome editing via nonhomologous end joining (NHEJ) or homology-directed repair (HDR) in mammalian cells, as well as generation of modified cell lines for downstream functional studies. to minimize off-target cleavage, we further describe a double-nicking strategy using the Cas9 nickase mutant with paired guide RNAs. This protocol provides experimentally derived guidelines for the selection of target sites, evaluation of cleavage efficiency and analysis of off-target activity. Beginning with target design, gene modifications can be achieved within as little as 1–2 weeks, and modified clonal cell lines can be derived within 2–3 weeks.
PMCID: PMC3969860  PMID: 24157548
4.  Fast Vesicle Transport Is Required for the Slow Axonal Transport of Synapsin 
The Journal of Neuroscience  2013;33(39):15362-15375.
Although it is known that cytosolic/soluble proteins synthesized in cell bodies are transported at much lower overall velocities than vesicles in fast axonal transport, the fundamental basis for this slow movement is unknown. Recently, we found that cytosolic proteins in axons of mouse cultured neurons are conveyed in a manner that superficially resembles diffusion, but with a slow anterograde bias that is energy- and motor-dependent (Scott et al., 2011). Here we show that slow axonal transport of synapsin, a prototypical member of this rate class, is dependent upon fast vesicle transport. Despite the distinct overall dynamics of slow and fast transport, experimentally induced and intrinsic variations in vesicle transport have analogous effects on slow transport of synapsin as well. Dynamic cotransport of vesicles and synapsin particles is also seen in axons, consistent with a model where higher-order assemblies of synapsin are conveyed by transient and probabilistic associations with vesicles moving in fast axonal transport. We posit that such dynamic associations generate the slow overall anterogradely biased flow of the population (“dynamic-recruitment model”). Our studies uncover the underlying kinetic basis for a classic cytosolic/soluble protein moving in slow axonal transport and reveal previously unknown links between slow and fast transport, offering a clearer conceptual picture of this curious phenomenon.
PMCID: PMC3782618  PMID: 24068803
5.  The Role of Lipolysis Stimulated Lipoprotein Receptor in Breast Cancer and Directing Breast Cancer Cell Behavior 
PLoS ONE  2014;9(3):e91747.
The claudin-low molecular subtype of breast cancer is of particular interest for clinically the majority of these tumors are poor prognosis, triple negative, invasive ductal carcinomas. Claudin-low tumors are characterized by cancer stem cell-like features and low expression of cell junction and adhesion proteins. Herein, we sought to define the role of lipolysis stimulated lipoprotein receptor (LSR) in breast cancer and cancer cell behavior as LSR was recently correlated with tumor-initiating features. We show that LSR was expressed in epithelium, endothelium, and stromal cells within the healthy breast tissue, as well as in tumor epithelium. In primary breast tumor bioposies, LSR expression was significantly correlated with invasive ductal carcinomas compared to invasive lobular carcinomas, as well as ERα positive tumors and breast cancer cell lines. LSR levels were significantly reduced in claudin-low breast cancer cell lines and functional studies illustrated that re-introduction of LSR into a claudin-low cell line suppressed the EMT phenotype and reduced individual cell migration. However, our data suggest that LSR may promote collective cell migration. Re-introduction of LSR in claudin-low breast cancer cell lines reestablished tight junction protein expression and correlated with transepithelial electrical resistance, thereby reverting claudin-low lines to other intrinsic molecular subtypes. Moreover, overexpression of LSR altered gene expression of pathways involved in transformation and tumorigenesis as well as enhanced proliferation and survival in anchorage independent conditions, highlighting that reestablishment of LSR signaling promotes aggressive/tumor initiating cell behaviors. Collectively, these data highlight a direct role for LSR in driving aggressive breast cancer behavior.
PMCID: PMC3956714  PMID: 24637461
6.  Development and optimization of an integrated PDMS based-microdialysis microchip electrophoresis device with on-chip derivatization for continuous monitoring of primary amines 
Electrophoresis  2013;34(6):895-902.
An all-PDMS on-line microdialysis-microchip electrophoresis with on-chip derivatization and electrophoretic separation for near real-time monitoring of primary amine-containing analytes is described. Each part of the chip was optimized separately, and the effect of each of the components on temporal resolution, lag time, and separation efficiency of the device was determined. Aspartate and glutamate were employed as test analytes. Derivatization was accomplished with naphthalene-2,3,-dicarboxyaldehyde/cyanide (NDA/CN−), and the separation was performed using a 15-cm serpentine channel. The analytes were detected using LIF detection.
PMCID: PMC3744098  PMID: 23335091
Aspartate; Cyanide; Glutamate; Microchip electrophoresis; Microdialysis; Naphthalene-2; 3-dicarboxaldehyde
7.  EULAR recommendations for the management of rheumatoid arthritis with synthetic and biological disease-modifying antirheumatic drugs: 2013 update 
Annals of the Rheumatic Diseases  2013;73(3):492-509.
In this article, the 2010 European League against Rheumatism (EULAR) recommendations for the management of rheumatoid arthritis (RA) with synthetic and biological disease-modifying antirheumatic drugs (sDMARDs and bDMARDs, respectively) have been updated. The 2013 update has been developed by an international task force, which based its decisions mostly on evidence from three systematic literature reviews (one each on sDMARDs, including glucocorticoids, bDMARDs and safety aspects of DMARD therapy); treatment strategies were also covered by the searches. The evidence presented was discussed and summarised by the experts in the course of a consensus finding and voting process. Levels of evidence and grades of recommendations were derived and levels of agreement (strengths of recommendations) were determined. Fourteen recommendations were developed (instead of 15 in 2010). Some of the 2010 recommendations were deleted, and others were amended or split. The recommendations cover general aspects, such as attainment of remission or low disease activity using a treat-to-target approach, and the need for shared decision-making between rheumatologists and patients. The more specific items relate to starting DMARD therapy using a conventional sDMARD (csDMARD) strategy in combination with glucocorticoids, followed by the addition of a bDMARD or another csDMARD strategy (after stratification by presence or absence of adverse risk factors) if the treatment target is not reached within 6 months (or improvement not seen at 3 months). Tumour necrosis factor inhibitors (adalimumab, certolizumab pegol, etanercept, golimumab, infliximab, biosimilars), abatacept, tocilizumab and, under certain circumstances, rituximab are essentially considered to have similar efficacy and safety. If the first bDMARD strategy fails, any other bDMARD may be used. The recommendations also address tofacitinib as a targeted sDMARD (tsDMARD), which is recommended, where licensed, after use of at least one bDMARD. Biosimilars are also addressed. These recommendations are intended to inform rheumatologists, patients, national rheumatology societies and other stakeholders about EULAR's most recent consensus on the management of RA with sDMARDs, glucocorticoids and bDMARDs. They are based on evidence and expert opinion and intended to improve outcome in patients with RA.
PMCID: PMC3933074  PMID: 24161836
Rheumatoid Arthritis; DMARDs (synthetic); DMARDs (biologic); Treatment; Early Rheumatoid Arthritis
8.  Optical Control of Mammalian Endogenous Transcription and Epigenetic States 
Nature  2013;500(7463):10.1038/nature12466.
The dynamic nature of gene expression enables cellular programming, homeostasis, and environmental adaptation in living systems. Dissection of causal gene functions in cellular and organismal processes therefore necessitates approaches that enable spatially and temporally precise modulation of gene expression. Recently, a variety of microbial and plant-derived light-sensitive proteins have been engineered as optogenetic actuators, enabling high precision spatiotemporal control of many cellular functions1-11. However, versatile and robust technologies that enable optical modulation of transcription in the mammalian endogenous genome remain elusive. Here, we describe the development of Light-Inducible Transcriptional Effectors (LITEs), an optogenetic two-hybrid system integrating the customizable TALE DNA-binding domain12-14 with the light-sensitive cryptochrome 2 protein and its interacting partner CIB1 from Arabidopsis thaliana. LITEs do not require additional exogenous chemical co-factors, are easily customized to target many endogenous genomic loci, and can be activated within minutes with reversibility3,4,6,7,15. LITEs can be packaged into viral vectors and genetically targeted to probe specific cell populations. We have applied this system in primary mouse neurons, as well as in the brain of awake mice in vivo to mediate reversible modulation of mammalian endogenous gene expression as well as targeted epigenetic chromatin modifications. The LITE system establishes a novel mode of optogenetic control of endogenous cellular processes and enables direct testing of the causal roles of genetic and epigenetic regulation in normal biological processes and disease states.
PMCID: PMC3856241  PMID: 23877069
9.  Gene Expression–Based Model Using Formalin-Fixed Paraffin-Embedded Biopsies Predicts Overall Survival in Advanced-Stage Classical Hodgkin Lymphoma 
Journal of Clinical Oncology  2012;31(6):692-700.
Our aim was to reliably identify patients with advanced-stage classical Hodgkin lymphoma (cHL) at increased risk of death by developing a robust predictor of overall survival (OS) using gene expression measured in routinely available formalin-fixed paraffin-embedded tissue (FFPET).
Expression levels of 259 genes, including those previously reported to be associated with outcome in cHL, were determined by digital expression profiling of pretreatment FFPET biopsies from 290 patients enrolled onto the E2496 Intergroup trial comparing doxorubicin, bleomycin, vinblastine, and dacarbazine (ABVD) and Stanford V regimens in locally extensive and advanced-stage cHL. A model for OS separating patients into low- and high-risk groups was produced using penalized Cox regression. The model was tested in an independent cohort of 78 patients enriched for treatment failure but otherwise similar to patients in a population-based registry of patients treated with ABVD. Weighted analysis methods generated unbiased estimates of predictor performance in the population-based registry.
A 23-gene outcome predictor was generated. The model identified a population at increased risk of death in the validation cohort. There was a 29% absolute difference in 5-year OS between the high- and low-risk groups (63% v 92%, respectively; log-rank P < .001; hazard ratio, 6.7; 95% CI, 2.6 to 17.4). The predictor was superior to the International Prognostic Score and CD68 immunohistochemistry in multivariate analyses.
A gene expression–based predictor, developed in and applicable to routinely available FFPET biopsies, identifies patients with advanced-stage cHL at increased risk of death when treated with standard-intensity up-front regimens.
PMCID: PMC3574267  PMID: 23182984
10.  Metabolic and Bactericidal Effects of Targeted Suppression of NadD and NadE Enzymes in Mycobacteria 
mBio  2014;5(1):e00747-13.
Mycobacterium tuberculosis remains a major cause of death due to the lack of treatment accessibility, HIV coinfection, and drug resistance. Development of new drugs targeting previously unexplored pathways is essential to shorten treatment time and eliminate persistent M. tuberculosis. A promising biochemical pathway which may be targeted to kill both replicating and nonreplicating M. tuberculosis is the biosynthesis of NAD(H), an essential cofactor in multiple reactions crucial for respiration, redox balance, and biosynthesis of major building blocks. NaMN adenylyltransferase (NadD) and NAD synthetase (NadE), the key enzymes of NAD biosynthesis, were selected as promising candidate drug targets for M. tuberculosis. Here we report for the first time kinetic characterization of the recombinant purified NadD enzyme, setting the stage for its structural analysis and inhibitor development. A protein knockdown approach was applied to validate bothNadD and NadE as target enzymes. Induced degradation of either target enzyme showed a strong bactericidal effect which coincided with anticipated changes in relative levels of NaMN and NaAD intermediates (substrates of NadD and NadE, respectively) and ultimate depletion of the NAD(H) pool. A metabolic catastrophe predicted as a likely result of NAD(H) deprivation of cellular metabolism was confirmed by 13C biosynthetic labeling followed by gas chromatography-mass spectrometry (GC-MS) analysis. A sharp suppression of metabolic flux was observed in multiple NAD(P)(H)-dependent pathways, including synthesis of many amino acids (serine, proline, aromatic amino acids) and fatty acids. Overall, these results provide strong validation of the essential NAD biosynthetic enzymes, NadD and NadE, as antimycobacterial drug targets.
To address the problems of M. tuberculosis drug resistance and persistence of tuberculosis, new classes of drug targets need to be explored. The biogenesis of NAD cofactors was selected for target validation because of their indispensable role in driving hundreds of biochemical transformations. We hypothesized that the disruption of NAD production in the cell via genetic suppression of the essential enzymes (NadD and NadE) involved in the last two steps of NAD biogenesis would lead to cell death, even under dormancy conditions. In this study, we confirmed the hypothesis using a protein knockdown approach in the model system of Mycobacterium smegmatis. We showed that induced proteolytic degradation of either target enzyme leads to depletion of the NAD cofactor pool, which suppresses metabolic flux through numerous NAD(P)-dependent pathways of central metabolism of carbon and energy production. Remarkably, bactericidal effect was observed even for nondividing bacteria cultivated under carbon starvation conditions.
PMCID: PMC3944813  PMID: 24549842
11.  ACPA-positive and ACPA-negative rheumatoid arthritis differ in their requirements for combination DMARDs and corticosteroids: secondary analysis of a randomized controlled trial 
UK guidelines recommend that all early active rheumatoid arthritis (RA) patients are offered combination disease-modifying antirheumatic drugs (DMARDs) and short-term corticosteroids. Anti-citrullinated protein antibody (ACPA)-positive and ACPA-negative RA may differ in their treatment responses. We used data from a randomized controlled trial - the Combination Anti-Rheumatic Drugs in Early RA (CARDERA) trial - to examine whether responses to intensive combination treatments in early RA differ by ACPA status.
The CARDERA trial randomized 467 early active RA patients to receive: (1) methotrexate, (2) methotrexate/ciclosporin, (3) methotrexate/prednisolone or (4) methotrexate/ciclosporin/prednisolone in a factorial-design. Patients were assessed every six months for two years. In this analysis we evaluated 431 patients with available ACPA status. To minimize multiple testing we used a mixed-effects repeated measures ANOVA model to test for an interaction between ACPA and treatment on mean changes from baseline for each outcome (Larsen, disease activity scores on a 28-joint count (DAS28), Health Assessment Questionnaire (HAQ), EuroQol, SF-36 physical component summary (PCS) and mental component summary (MCS) scores). When a significant interaction was present, mean changes in outcomes were compared by treatment group at each time point using t-tests stratified by ACPA status. Odds ratios (ORs) for the onset of new erosions with treatment were calculated stratified by ACPA.
ACPA status influenced the need for combination treatments to reduce radiological progression. ACPA-positive patients had significant reductions in Larsen score progression with all treatments. ACPA-positive patients receiving triple therapy had the greatest benefits: two-year mean Larsen score increases comprised 3.66 (95% confidence interval (CI) 2.27 to 5.05) with triple therapy and 9.58 (95% CI 6.76 to 12.39) with monotherapy; OR for new erosions with triple therapy versus monotherapy was 0.32 (95% CI 0.14 to 0.72; P = 0.003). ACPA-negative patients had minimal radiological progression irrespective of treatment. Corticosteroid’s impact on improving DAS28/PCS scores was confined to ACPA-positive RA.
ACPA status influences the need for combination DMARDs and high-dose tapering corticosteroids in early RA. In CARDERA, combination therapy was only required to prevent radiological progression in ACPA-positive patients; corticosteroids only provided significant disease activity and physical health improvements in ACPA-positive disease. This suggests ACPA is an important biomarker for guiding treatment decisions in early RA.
Trial registration
Current Controlled Trials ISRCTN32484878
PMCID: PMC3979097  PMID: 24433430
12.  Prey field switching based on preferential behaviour can induce Lévy flights 
Using the foraging movements of an insectivorous bat, Myotis mystacinus, we describe temporal switching of foraging behaviour in response to resource availability. These observations conform to predictions of optimized search under the Lévy flight paradigm. However, we suggest that this occurs as a result of a preference behaviour and knowledge of resource distribution. Preferential behaviour and knowledge of a familiar area generate distinct movement patterns as resource availability changes on short temporal scales. The behavioural response of predators to changes in prey fields can elicit different functional responses, which are considered to be central in the development of stable predator–prey communities. Recognizing how the foraging movements of an animal relate to environmental conditions also elucidates the evolution of optimized search and the prevalence of discrete strategies in natural systems. Applying techniques that use changes in the frequency distribution of movements facilitates exploration of the processes that underpin behavioural changes.
PMCID: PMC3565776  PMID: 23054951
Lévy flight; functional responses; bats; areoecology
13.  ‘Transient’ Genetic Suppression Facilitates Generation of Hexose Transporter Null Mutants in Leishmania mexicana 
Molecular microbiology  2012;87(2):412-429.
The genome of Leishmania mexicana encompasses a cluster of three glucose transporter genes designated LmxGT1, LmxGT2, and LmxGT3. Functional and genetic studies of a cluster null mutant (Δlmxgt1-3) have dissected the roles of these proteins in Leishmania metabolism and virulence. However, null mutants were recovered at very low frequency, and comparative genome hybridizations revealed that Δlmxgt1-3 mutants contained a linear extrachromosomal 40 kb amplification of a region on chromosome 29 not amplified in WT parasites. These data suggested a model where this 29–40k amplicon encoded a second site suppressor contributing to parasite survival in the absence of GT1-3 function. To test this, we quantified the frequency of recovery of knockouts in the presence of individual overexpressed ORFs covering the 29–40k amplicon. The data mapped the suppressor activity to PIFTC3, encoding a component of the intraflagellar transport pathway. We discuss possible models by which PIFTC3 might act to facilitate loss of GTs specifically. Surprisingly, by plasmid segregation we showed that continued PIFTC3 overexpression was not required for Δlmxgt1-3 viability. These studies provide the first evidence that genetic suppression can occur by providing critical biological functions transiently. This novel form of genetic suppression may extend to other genes, pathways and organisms.
PMCID: PMC3545093  PMID: 23170981
14.  32 species validation of a new Illumina paired-end approach for the development of microsatellites 
PLoS ONE  2013;8(11):e81853.
Development and optimization of novel species-specific microsatellites, or simple sequence repeats (SSRs) remains an important step for studies in ecology, evolution, and behavior. Numerous approaches exist for identifying new SSRs that vary widely in terms of both time and cost investments. A recent approach of using paired-end Illumina sequence data in conjunction with the bioinformatics pipeline, PAL_FINDER, has the potential to substantially reduce the cost and labor investment while also improving efficiency. However, it does not appear that the approach has been widely adopted, perhaps due to concerns over its broad applicability across taxa. Therefore, to validate the utility of the approach we developed SSRs for 32 species representing 30 families, 25 orders, 11 classes, and six phyla and optimized SSRs for 13 of the species. Overall the IPE method worked extremely well and we identified 1000s of SSRs for all species (mean = 128,485), with 17% of loci being potentially amplifiable loci, and 25% of these met our most stringent criteria designed to that avoid SSRs associated with repetitive elements. Approximately 61% of screened primers yielded strong amplification of a single locus.
PMCID: PMC3842982  PMID: 24312368
15.  Role for endothelial N-glycan mannose residues in monocyte recruitment during atherogenesis 
Arteriosclerosis, thrombosis, and vascular biology  2012;32(8):10.1161/ATVBAHA.112.253203.
Up-regulated expression of endothelial adhesion molecules and subsequent binding to cognate monocytic receptors is an established paradigm in atherosclerosis. However, these proteins are the scaffolds with their post-translational modification with sugars providing the actual ligands. We showed recently that TNFα increased hypoglycosylated (mannose rich) N-glycans on the endothelial surface. In this study our aim was to determine if i) hypoglycosylated N-glycans are upregulated by pro-atherogenic stimuli (oscillatory flow) in vitro and in vivo, and ii) whether mannose residues on hypoglycosylated endothelial N-glycans mediate monocyte rolling and adhesion.
Methods and result
Staining with the mannose specific lectins ConA and LCA was increased in human aortic endothelial cells exposed to oscillatory shear or TNFα, and at sites of plaque development and progression in both mice and human vessels. Increasing surface N-linked mannose by inhibiting N-glycan processing, potentiated monocyte adhesion under flow during TNFα stimulation. Conversely, enzymatic removal of high-mannose N-glycans, or masking mannose residues with lectins, significantly decreased monocyte adhesion under flow. These effects occurred without altering induced expression of adhesion molecule proteins.
Hypoglycosylated (high mannose) N-glycans are present on the endothelial cell surface at sites of early human lesion development and are novel effectors of monocyte adhesion during atherogenesis.
PMCID: PMC3831355  PMID: 22723438
mannose; inflammation; vascular; adhesion; glycobiology
16.  The effect of varying acidity on helicobacter pylori growth and the bactericidal efficacy of ampicillin 
Penicillins inhibit cell wall synthesis; therefore, H. pylori must be dividing for this class of antibiotics to be effective in eradication therapy. Identifying growth responses to varying medium pH may allow design of more effective treatment regimens.
To determine the effect of acidity on bacterial growth and the bactericidal efficacy of ampicillin.
H. pylori were incubated in dialysis chambers suspended in 1.5L of media at various pHs with 5mM urea, with or without ampicillin, for 4, 8 or 16 hours, thus mimicking unbuffered gastric juice. Changes in gene expression, viability and survival were determined.
At pH 3.0, but not at pH 4.5 or 7.4, there was decreased expression of ~400 genes, including many cell envelope biosynthesis, cell division and penicillin-binding protein genes. Ampicillin was bactericidal at pH 4.5 and 7.4 but not at pH 3.0.
Ampicillin is bactericidal at pH 4.5 and 7.4, but not at pH 3.0, due to decreased expression of cell envelope and division genes with loss of cell division at pH 3.0. Therefore, at pH 3.0, the likely pH at the gastric surface, the bacteria are non-dividing and persist with ampicillin treatment. A more effective inhibitor of acid secretion that maintains gastric pH near neutrality for 24 hours/day should enhance the efficacy of amoxicillin, improving triple therapy and likely even allowing dual amoxicillin based therapy for H. pylori eradication.
PMCID: PMC3474890  PMID: 23009227
Helicobacter pylori; medium pH; bacterial growth; ampicillin/amoxicillin; gastric acid inhibition; proton pump inhibitor
17.  Glutamine-fueled mitochondrial metabolism is decoupled from glycolysis in melanoma 
Pigment cell & melanoma research  2012;25(6):732-739.
In this perspective, we revise the historic notion that cancer is a disease of mitochondria. We summarize recent findings on the function and rewiring of central carbon metabolism in melanoma. Metabolic profiling studies using stable isotope tracers show that glycolysis is decoupled from the tricarboxylic acid (TCA) cycle. This decoupling is not ‘dysfunction’ but rather an alternate wiring required by tumor cells to remain metabolically versatile. In large part, this requirement is met by glutamine feeding the TCA cycle as an alternative source of carbon. Glutamine is also used in non-conventional ways, like traveling in reverse through the TCA flux to feed fatty acid biosynthesis. The biosynthetic networks linked with non-essential amino acids alanine, serine, arginine, and proline are also significantly impacted by the use of glutamine as an alternate carbon source.
PMCID: PMC3639292  PMID: 22846158
metabolism; mitochondria; glutamine; systems biology; NMR
18.  Leishmania salvage and remodelling of host sphingolipids in amastigote survival and acidocalcisome biogenesis 
Molecular microbiology  2005;55(5):1566-1578.
Sphingolipids (SLs) play essential roles in most eukaryotes, but in the trypanosomatid protozoan Leishmania major their functions differ significantly. Previously we showed that null mutants defective in de novo sphingoid base synthesis (spt2−) lacked SLs but grew well and retained lipid rafts while replicating as promastigotes in vitro. However, they experienced catastrophic defects in membrane trafficking on entry into stationary phase, and failed to differentiate to the infective metacyclic form. Here we showed this mutant retained the ability to enter macrophages silently and inhibit activation, although as expected most parasites were destroyed. However, in mouse infections, after a delay rapidly progressive lesions appeared, and purified amastigotes were fully virulent to macrophages and mice. Mass spectrometry of spt2− amastigote lipids revealed the presence of high levels of parasite-specific inositol phosphorylceramides (IPCs) not synthesized by the mammalian hosts. Inhibitor studies showed that salvage occurs at the level of complex SLs, suggesting that parasites carry out ‘headgroup’ remodelling. Additionally, we describe a new defect of the spt2− promastigotes involving ‘empty’ acidocalcisomes (ACs), which may point to the origin of this organelle from the lysosome-related organelle/multivesicular body biogenesis pathway. However, ACs in spt2− amastigotes appeared quantitatively and morphologically normal. Thus salvage of SLs and other molecules by intracellular amastigotes play key roles in AC biogenesis and parasite survival in the host.
PMCID: PMC3803142  PMID: 15720561
19.  Treatment of type 2 diabetes in chronic kidney disease: a case for linagliptin in the treatment of diabetes in severe renal impairment 
Diabetes is the leading cause of chronic kidney disease, and the prevalence of both diseases is rising worldwide. Treatment of type 2 diabetes is difficult in patients with chronic kidney disease because most oral antidiabetic agents are affected by renal function and their use may be contraindicated in this patient population. Antidiabetic agents that can be used in patients with type 2 diabetes and declining renal function are needed. Incretin-based therapies, such as dipeptidyl peptidase-4 inhibitors, are a recent therapeutic class of glucose-lowering agents that may offer an effective treatment option in patients with chronic kidney disease. Within the dipeptidyl peptidase-4 class, linagliptin has a unique profile with a primarily nonrenal route of elimination, requiring no dose adjustment in patients with chronic kidney disease. This communication summarizes the findings of a 1-year, randomized, double-blind, placebo-controlled study demonstrating the favorable safety and efficacy profile of linagliptin in patients with type 2 diabetes and severe renal impairment.
PMCID: PMC3795009  PMID: 24124385
renal impairment; incretin; DPP-4 inhibitor; type 2 diabetes
20.  Concurrent Expression of MYC and BCL2 in Diffuse Large B-Cell Lymphoma Treated With Rituximab Plus Cyclophosphamide, Doxorubicin, Vincristine, and Prednisone 
Journal of Clinical Oncology  2012;30(28):3452-3459.
Diffuse large B-cell lymphoma (DLBCL) is curable in 60% of patients treated with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP). MYC translocations, with or without BCL2 translocations, have been associated with inferior survival in DLBCL. We investigated whether expression of MYC protein, with or without BCL2 protein expression, could risk-stratify patients at diagnosis.
Patients and Methods
We determined the correlation between presence of MYC and BCL2 proteins by immunohistochemistry (IHC) with survival in two independent cohorts of patients with DLBCL treated with R-CHOP. We further determined if MYC protein expression correlated with high MYC mRNA and/or presence of MYC translocation.
In the training cohort (n = 167), MYC and BCL2 proteins were detected in 29% and 44% of patients, respectively. Concurrent expression (MYC positive/BCL2 positive) was present in 21% of patients. MYC protein correlated with presence of high MYC mRNA and MYC translocation (both P < .001), but the latter was less frequent (both 11%). MYC protein expression was only associated with inferior overall and progression-free survival when BCL2 protein was coexpressed (P < .001). Importantly, the poor prognostic effect of MYC positive/BCL2 positive was validated in an independent cohort of 140 patients with DLBCL and remained significant (P < .05) after adjusting for presence of high-risk features in a multivariable model that included elevated international prognostic index score, activated B-cell molecular subtype, and presence of concurrent MYC and BCL2 translocations.
Assessment of MYC and BCL2 expression by IHC represents a robust, rapid, and inexpensive approach to risk-stratify patients with DLBCL at diagnosis.
PMCID: PMC3454768  PMID: 22851565
21.  Clinical Human Papillomavirus Detection Forecasts Cervical Cancer Risk in Women Over 18 Years of Follow-Up 
Journal of Clinical Oncology  2012;30(25):3044-3050.
To describe the long-term (≥ 10 years) benefits of clinical human papillomavirus (HPV) DNA testing for cervical precancer and cancer risk prediction.
Cervicovaginal lavages collected from 19,512 women attending a health maintenance program were retrospectively tested for HPV using a clinical test. HPV positives were tested for HPV16 and HPV18 individually using a research test. A Papanicolaou (Pap) result classified as atypical squamous cells of undetermined significance (ASC-US) or more severe was considered abnormal. Women underwent follow-up prospectively with routine annual Pap testing up to 18 years. Cumulative incidence rates (CIRs) of ≥ grade 3 cervical intraepithelial neoplasia (CIN3+) or cancer for enrollment test results were calculated.
A baseline negative HPV test provided greater reassurance against CIN3+ over the 18-year follow-up than a normal Pap (CIR, 0.90% v 1.27%). Although both baseline Pap and HPV tests predicted who would develop CIN3+ within the first 2 years of follow-up, only HPV testing predicted who would develop CIN3+ 10 to 18 years later (P = .004). HPV16- and HPV18-positive women with normal Pap were at elevated risk of CIN3+ compared with other HPV-positive women with normal Pap and were at similar risk of CIN3+ compared with women with a low-grade squamous intraepithelial Pap.
HPV testing to rule out cervical disease followed by Pap testing and possibly combined with the detection of HPV16 and HPV18 among HPV positives to identify those at immediate risk of CIN3+ would be an efficient algorithm for cervical cancer screening, especially in women age 30 years or older.
PMCID: PMC3732003  PMID: 22851570
22.  The Prevalence and Characteristics of Dual PharmD/MPH Programs Offered at US Colleges and Schools of Pharmacy 
Objective. To assess the prevalence and characteristics of curriculum in dual doctor of pharmacy (PharmD)/master of public health (MPH) degree programs offered by US pharmacy programs.
Methods. An 18-item survey instrument was developed and distributed online to faculty members at US colleges and schools of pharmacy.
Results. Of the 110 colleges and schools that responded, 23 (21%) offered a PharmD/MPH degree. Common characteristics of these 23 programs included current PharmD program structure (3 + 1 year), early curricular recruitment, small enrollment, and interdisciplinary coursework occurring online and in the classroom. The impact of the dual degree on the curriculum and longevity of the dual-degree programs varied. About 55% of responding programs without a formal dual-degree program reported that additional public health training was available.
Conclusion. Twenty-one percent of colleges and schools of pharmacy offer a combined PharmD/MPH dual degree. Most programs required an additional 1 or 2 semesters to complete both degrees.
PMCID: PMC3748297  PMID: 23966719
pharmacy education; public health; masters of public health; dual degree
23.  Heterogenic Endothelial Responses to Inflammation: Role for Differential N‐Glycosylation and Vascular Bed of Origin 
Endothelial cell responses during inflammation are heterogeneous and key for selectivity in how leukocytes hone in on specific sites and why vascular diseases are highly bed specific. However, mechanisms for this specificity remain unclear.
Methods and Results
Here, we exposed human endothelial cells isolated from 5 systemic arterial beds from 1 donor (to overcome donor‐to‐donor genetic/epigenetic differences), the umbilical vein, and pulmonary microvasculature to TNF‐α, LPS, and IL‐1β and assessed acute (ERK1/2 and p65) and chronic (ICAM‐1, VCAM‐1 total and surface expression) signaling responses and assessed changes in surface N‐glycans and monocyte adhesion. Significant diversity in responses was evident by disparate changes in ERK1/2 and p65 NF‐κB phosphorylation, which varied up to 5‐fold between different cells and in temporal and magnitude differences in ICAM‐1 and VCAM‐1 expression (maximal VCAM‐1 induction typically being observed by 4 hours, whereas ICAM‐1 expression was increased further at 24 hours relative to 4 hours). N‐glycan profiles both basally and with stimulation were also bed specific, with hypoglycosylated N‐glycans correlating with increased THP‐1 monocyte adhesion. Differences in surface N‐glycan expression tracked with dynamic up‐ or downregulation of α‐mannosidase activity during inflammation.
These results demonstrate a critical role for the vascular bed of origin in controlling endothelial responses and function to inflammatory stimuli and suggest that bed‐specific expression of N‐linked sugars may provide a signature for select leukocyte recruitment.
PMCID: PMC3828811  PMID: 23900214
heterogeneity; mannose; N‐glycans
24.  B Cells With High Side Scatter Parameter by Flow Cytometry Correlate With Inferior Survival in Diffuse Large B-Cell Lymphoma 
Despite advances in the understanding of diffuse large B-cell lymphoma (DLBCL) biology, only the clinically based International Prognostic Index (IPI) is used routinely for risk stratification at diagnosis. To find novel prognostic markers, we analyzed flow cytometric data from 229 diagnostic DLBCL samples using an automated multiparameter data analysis approach developed in our laboratory. By using the developed automated data analysis pipeline, we identified 71 of 229 cases as having more than 35% B cells with a high side scatter (SSC) profile, a parameter reflecting internal cellular complexity. This high SSC B-cell feature was associated with inferior overall and progression-free survival (P = .001 and P = .01, respectively) and remained a significant predictor of overall survival in multivariate Cox regression analysis (IPI, P = .001; high SSC, P = .004; rituximab, P = .53).
This study suggests that high SSC among B cells may serve as a useful biomarker to identify patients with DLBCL at high risk for relapse. This is of particular interest because this biomarker is readily available in most clinical laboratories without significant alteration to existing routine diagnostic strategies or incurring additional costs.
PMCID: PMC3718075  PMID: 22523221
Side scatter; Flow cytometry; Diffuse large B-cell lymphoma; Lymphoma; Survival
25.  Current perspectives on tocilizumab for the treatment of rheumatoid arthritis: a review 
Rheumatoid arthritis (RA) remains a major clinical problem with many patients having continuing systemic inflammatory disease resulting in progressive erosive damage and high levels of disability. A range of pro-inflammatory cytokines including tumor necrosis factor (TNF), interleukin (IL)-1 and IL-6 are involved in RA pathogenesis; these cytokines can be specifically inhibited by biological agents. Tocilizumab (TCZ) is a recombinant humanized anti-IL-6 receptor monoclonal antibody, administered monthly by intravenous infusion that prevents IL-6 signal transduction. There is strong evidence that it is both clinically efficacious and cost-effective. There have been several key clinical trials evaluating the safety and efficacy of TCZ in RA patients. We review five Phase II trials and seven Phase III trials enrolling a total of 626 and 5268 RA patients respectively. The American College of Rheumatology (ACR) response criteria were used as the primary or secondary outcome measure in all trials. Overall these trials demonstrated that TCZ was effective in the treatment of RA in a number of patient groups, including those with an inadequate response to methotrexate (MTX) or TNF inhibition. TCZ use, both as monotherapy and in combination with MTX, improved the signs and symptoms of RA within several weeks of commencing treatment. Additionally, TCZ was shown to reduce radiological disease progression and improve physical function, both as monotherapy and in combination with MTX. A 5-year extension study demonstrated that TCZ sustained good long-term efficacy and safety profiles. TCZ was generally well tolerated. Although its use increased the risk of an adverse event, these were usually mild to moderate in severity and treatment did not increase the risk of a serious adverse event in comparison to controls. Due to moderate increases in serum levels of total cholesterol, triglycerides, high-density lipoproteins and serum transaminases seen in those patients treated with TCZ, as well as severe neutropenia in some, regular blood monitoring of full blood count, liver function and lipids is recommended. Given its clinical efficacy in the treatment of RA, TCZ may be beneficial in the treatment of other autoimmune diseases where IL-6 plays a role in the inflammatory cascade.
PMCID: PMC3706382  PMID: 23869169
tocilizumab; rheumatoid arthritis; review; IL-6

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