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1.  Positive allosteric modulation of GABAB receptors ameliorates sensorimotor gating in rodent models 
CNS neuroscience & therapeutics  2014;20(7):679-684.
Converging evidence points to the involvement of γ-amino-butyric acid B receptors (GABABRs) in the regulation of information processing. We previously showed that GABABR agonists exhibit antipsychotic-like properties in rodent models of sensorimotor gating deficits, as measured by the prepulse inhibition (PPI) of the acoustic startle reflex. The therapeutic potential of these agents, however, is limited by their neuromuscular side effects; thus, in the present study we analyzed whether rac-BHFF, a potent GABABR positive allosteric modulator (PAM), could counter spontaneous and pharmacologically induced PPI deficits across various rodent models.
We tested the antipsychotic effects of rac-BHFF on the PPI deficits caused by the N-methyl-D-aspartate glutamate receptor antagonist dizocilpine, in Sprague-Dawley rats and C57BL/6 mice. Furthermore, we verified whether rac-BHFF ameliorated the spontaneous PPI impairments in DBA/2J mice.
rac-BHFF dose-dependently countered the PPI deficits across all three models, in a fashion akin to the GABABR agonist baclofen and the atypical antipsychotic clozapine; in contrast with these compounds, however, rac-BHFF did not affect startle magnitude.
The present data further support the implication of GABABRs in the modulation of sensorimotor gating, and point to their PAMs as a novel promising tool for antipsychotic treatment, with fewer side effects than GABABR agonists.
PMCID: PMC4062587  PMID: 24703381
GABAB receptors; prepulse inhibition of the startle; sensorimotor gating; schizophrenia; NMDA receptors; DBA/2J mice
2.  Stromal Transcriptional Profiles Reveal Hierarchies of Anatomical Site, Serum Response and Disease and Identify Disease Specific Pathways 
PLoS ONE  2015;10(3):e0120917.
Synovial fibroblasts in persistent inflammatory arthritis have been suggested to have parallels with cancer growth and wound healing, both of which involve a stereotypical serum response programme. We tested the hypothesis that a serum response programme can be used to classify diseased tissues, and investigated the serum response programme in fibroblasts from multiple anatomical sites and two diseases. To test our hypothesis we utilized a bioinformatics approach to explore a publicly available microarray dataset including rheumatoid arthritis (RA), osteoarthritis (OA) and normal synovial tissue, then extended those findings in a new microarray dataset representing matched synovial, bone marrow and skin fibroblasts cultured from RA and OA patients undergoing arthroplasty. The classical fibroblast serum response programme discretely classified RA, OA and normal synovial tissues. Analysis of low and high serum treated fibroblast microarray data revealed a hierarchy of control, with anatomical site the most powerful classifier followed by response to serum and then disease. In contrast to skin and bone marrow fibroblasts, exposure of synovial fibroblasts to serum led to convergence of RA and OA expression profiles. Pathway analysis revealed three inter-linked gene networks characterising OA synovial fibroblasts: Cell remodelling through insulin-like growth factors, differentiation and angiogenesis through _3 integrin, and regulation of apoptosis through CD44. We have demonstrated that Fibroblast serum response signatures define disease at the tissue level, and that an OA specific, serum dependent repression of genes involved in cell adhesion, extracellular matrix remodelling and apoptosis is a critical discriminator between cultured OA and RA synovial fibroblasts.
PMCID: PMC4373951  PMID: 25807374
3.  Heat Shock Protein 22 (Hsp22) Regulates Oxidative Phosphorylation upon Its Mitochondrial Translocation with the Inducible Nitric Oxide Synthase in Mammalian Heart 
PLoS ONE  2015;10(3):e0119537.
Stress-inducible heat shock protein 22 (Hsp22) confers protection against ischemia through induction of the inducible isoform of nitric oxide synthase (iNOS). Hsp22 overexpression in vivo stimulates cardiac mitochondrial respiration, whereas Hsp22 deletion in vivo significantly reduces respiration. We hypothesized that Hsp22-mediated regulation of mitochondrial function is dependent upon its mitochondrial translocation together with iNOS.
Methods and Results
Adenoviruses harboring either the full coding sequence of Hsp22 (Ad-WT-Hsp22) or a mutant lacking a N-terminal 20 amino acid putative mitochondrial localization sequence (Ad-N20-Hsp22) were generated, and infected in rat neonatal cardiomyocytes. Compared to β-Gal control, WT-Hsp22 accumulated in mitochondria by 2.5 fold (P<0.05) and increased oxygen consumption rates by 2-fold (P<0.01). This latter effect was abolished upon addition of the selective iNOS inhibitor, 1400W. Ad-WT-Hsp22 significantly increased global iNOS expression by about 2.5-fold (P<0.01), and also increased iNOS mitochondrial localization by 4.5 fold vs β-gal (P<0.05). Upon comparable overexpression, the N20-Hsp22 mutant did not show significant mitochondrial translocation or stimulation of mitochondrial respiration. Moreover, although N20-Hsp22 did increase global iNOS expression by 4.6-fold, it did not promote iNOS mitochondrial translocation.
Translocation of both Hsp22 and iNOS to the mitochondria is necessary for Hsp22-mediated stimulation of oxidative phosphorylation.
PMCID: PMC4352051  PMID: 25746286
4.  Reliable Encoding of Stimulus Intensities Within Random Sequences of Intracellular Ca2+ Spikes 
Science signaling  2014;7(331):ra59.
Ca2+ is a ubiquitous intracellular messenger that regulates diverse cellular activities. Extracellular stimuli often evoke sequences of intracellular Ca2+ spikes, and spike frequency may encode stimulus intensity. However, the timing of spikes within a cell is random because each interspike interval has a large stochastic component. In HEK293 (human embryonic kidney 293) cells and rat primary hepatocytes, we found that the average interspike interval also varied between individual cells. To evaluate how individual cells reliably encoded stimuli when Ca2+ spikes exhibited such unpredictability, we combined Ca2+ imaging of single cells with mathematical analyses of the Ca2+ spikes evoked by receptors that stimulate formation of inositol 1,4,5-trisphosphate (IP3). This analysis revealed that signal-to-noise ratios were improved by slow recovery from feedback inhibition of Ca2+ spiking operating at the whole-cell level and that they were robust against perturbations of the signaling pathway. Despite variability in the frequency of Ca2+ spikes between cells, steps in stimulus intensity caused the stochastic period of the interspike interval to change by the same factor in all cells. These fold changes reliably encoded changes in stimulus intensity, and they resulted in an exponential dependence of average interspike interval on stimulation strength. We conclude that Ca2+ spikes enable reliable signaling in a cell population despite randomness and cell-to-cell variability, because global feedback reduces noise, and changes in stimulus intensity are represented by fold changes in the stochastic period of the interspike interval.
PMCID: PMC4092318  PMID: 24962706
5.  Dietary Sutherlandia and Elderberry Mitigate Cerebral Ischemia-Induced Neuronal Damage and Attenuate p47phox and Phospho-ERK1/2 Expression in Microglial Cells 
ASN NEURO  2014;6(6):1759091414554946.
Sutherlandia (Sutherlandia frutescens) and elderberry (Sambucus spp.) are used to promote health and for treatment of a number of ailments. Although studies with cultured cells have demonstrated antioxidative and anti-inflammatory properties of these botanicals, little is known about their ability to mitigate brain injury. In this study, C57BL/6 J male mice were fed AIN93G diets without or with Sutherlandia or American elderberry for 2 months prior to a 30-min global cerebral ischemia induced by occlusion of the bilateral common carotid arteries (BCCAs), followed by reperfusion for 3 days. Accelerating rotarod assessment at 24 h after BCCA occlusion showed amelioration of sensorimotor impairment in the mice fed the supplemented diets as compared with the ischemic mice fed the control diet. Quantitative digital pathology assessment of brain slides stained with cresyl violet at 3 days after ischemia/reperfusion (I/R) revealed significant reduction in neuronal cell death in both dietary groups. Immunohistochemical staining for ionized calcium-binding adapter molecule-1 demonstrated pronounced activation of microglia in the hippocampus and striatum in the ischemic brains 3 days after I/R, and microglial activation was significantly reduced in animals fed supplemented diets. Mitigation of microglial activation by the supplements was further supported by the decrease in expression of p47phox, a cytosolic subunit of NADPH oxidase, and phospho-ERK1/2, a mitogen-activated protein kinase known to mediate a number of cytoplasmic processes including oxidative stress and neuroinflammatory responses. These results demonstrate neuroprotective effect of Sutherlandia and American elderberry botanicals against oxidative and inflammatory responses to cerebral I/R.
PMCID: PMC4271764
botanical diet; global cerebral ischemia; microglia; oxidative stress; p47phox; phospho-ERK1/2
6.  Dietary Sutherlandia and Elderberry Mitigate Cerebral Ischemia-Induced Neuronal Damage and Attenuate p47phox and Phospho-ERK1/2 Expression in Microglial Cells 
ASN NEURO  2014;6(6):1759091414554946.
Sutherlandia (Sutherlandia frutescens) and elderberry (Sambucus spp.) are used to promote health and for treatment of a number of ailments. Although studies with cultured cells have demonstrated antioxidative and anti-inflammatory properties of these botanicals, little is known about their ability to mitigate brain injury. In this study, C57BL/6 J male mice were fed AIN93G diets without or with Sutherlandia or American elderberry for 2 months prior to a 30-min global cerebral ischemia induced by occlusion of the bilateral common carotid arteries (BCCAs), followed by reperfusion for 3 days. Accelerating rotarod assessment at 24 h after BCCA occlusion showed amelioration of sensorimotor impairment in the mice fed the supplemented diets as compared with the ischemic mice fed the control diet. Quantitative digital pathology assessment of brain slides stained with cresyl violet at 3 days after ischemia/reperfusion (I/R) revealed significant reduction in neuronal cell death in both dietary groups. Immunohistochemical staining for ionized calcium-binding adapter molecule-1 demonstrated pronounced activation of microglia in the hippocampus and striatum in the ischemic brains 3 days after I/R, and microglial activation was significantly reduced in animals fed supplemented diets. Mitigation of microglial activation by the supplements was further supported by the decrease in expression of p47phox, a cytosolic subunit of NADPH oxidase, and phospho-ERK1/2, a mitogen-activated protein kinase known to mediate a number of cytoplasmic processes including oxidative stress and neuroinflammatory responses. These results demonstrate neuroprotective effect of Sutherlandia and American elderberry botanicals against oxidative and inflammatory responses to cerebral I/R.
PMCID: PMC4357609
botanical diet; global cerebral ischemia; microglia; oxidative stress; p47phox; phospho-ERK1/2
7.  Alcohol and tobacco consumption affects bacterial richness in oral cavity mucosa biofilms 
BMC Microbiology  2014;14:250.
Today there are more than 2 billion alcohol users and about 1.3 billion tobacco users worldwide. The chronic and heavy use of these two substances is at the heart of numerous diseases and may wreak havoc on the human oral microbiome. This study delves into the changes that alcohol and tobacco may cause on biofilms of the human oral microbiome. To do so, we used swabs to sample the oral biofilm of 22 subjects; including 9 control-individuals with no or very low consumption of alcohol and no consumption of tobacco, 7 who were chronic and heavy users of both substances and 6 active smokers that reported no significant alcohol consumption. DNA was extracted from swabs and the V1 region of the 16S rRNA gene was PCR amplified and sequenced using the Ion Torrent PGM platform, generating 3.7 million high quality reads. DNA sequences were clustered and OTUs were assigned using the ARB SILVA database and Qiime.
We found no differences in species diversity and evenness among the groups. However, we found a significant decrease in species richness in only smokers and in smokers/drinkers when compared to controls. We found that Neisseria abundance was significantly decreased in both groups when compared to controls. Smokers had significant increases in Prevotella and Capnocytophaga and reductions in Granulicatella, Staphylococcus, Peptostreptococcus and Gemella when compared to the two other groups. Controls showed higher abundance of Aggregibacter, whilst smokers/drinkers had lower abundances of Fusobacteria. Samples from only smokers clustered closer together than to controls and smokers/drinkers, and also had a significant reduction in inter-group dissimilarity distances, indicating a more homogenous group than controls.
Our results indicate that the continued use of tobacco or alcohol plus tobacco significantly reduces bacterial richness, which apparently leads to a reduction in inter-group variability, turning the respective biofilms into a more homogenous microenvironment in terms of bacterial community composition, with possible consequences for human oral diseases.
Electronic supplementary material
The online version of this article (doi:10.1186/s12866-014-0250-2) contains supplementary material, which is available to authorized users.
PMCID: PMC4186948  PMID: 25278091
Alcohol; Tobacco; Microbiome; Oral cavity; 16S rRNA
8.  Survival in Out-of-Hospital Cardiac Arrests with Initial Asystole or Pulseless Electrical Activity and Subsequent Shockable Rhythms 
Resuscitation  2013;84(9):1261-1266.
Non-shockable arrest rhythms (Pulseless Electrical Activity and Asystole) represent an increasing proportion of reported cases of out-of-hospital cardiac arrest (OHCA). The prognostic significance of conversion from non-shockable to shockable rhythms during the course of resuscitation remains unclear.
To evaluate whether out-of-hospital cardiac arrest survival with initially non-shockable arrest rhythms is improved with subsequent conversion to shockable rhythms.
Secondary analysis of data in Epistry – Cardiac Arrest, an epidemiologic registry maintained by the Resuscitation Outcomes Consortium (ROC). This analysis includes OHCA events from December 1, 2005 through May 31, 2007 contributed by six US and two Canadian sites. For all EMS-treated adult (18 and older) cardiac arrest patients who presented with non-shockable cardiac arrest, we compared survival to hospital discharge between patients who did develop a shockable rhythm and those who did not based on receipt of subsequent defibrillation. Missing data were handled using multiple imputation. Multivariable logistic regression was used to adjust for potentially confounding variables.
A total of 6,556 EMS treated adult cardiac arrest cases presented in non-shockable rhythms. Survival to discharge in patients who converted to a shockable rhythm was 2.77% while survival in those who did not was 2.72% (p = 0.92). After adjusting for confounders, conversion to a shockable rhythm was not associated with improved survival (OR 0.88, 95% CI: 0.60–1.30).
For OHCA patients presenting in PEA/Asystole, survival to hospital discharge was not associated with conversion to a shockable rhythm during EMS resuscitation efforts.
PMCID: PMC3947599  PMID: 23454257
Out-of-Hospital; Cardiac Arrest; Non-shockable Rhythms; Survival
9.  Uterine artery embolization immediately preceding laparoscopic myomectomy 
To determine whether performing uterine artery embolization (UAE) immediately before laparoscopic myomectomy can facilitate a minimally invasive surgical approach for larger uterine fibroids.
In a retrospective case–control study, laparoscopic myomectomy with and without preoperative UAE was examined. Data were analyzed from 26 laparoscopic myomectomies performed by a single surgeon at Northwestern University Feinberg School of Medicine between 2004 and 2010. Controls were matched for age, calendar year, surgeon, and number of fibroids removed. Surgical outcomes included preoperative clinical uterine size, operative time, operative blood loss, and postoperative myoma specimen weight. Data were analyzed via 2-tailed Student t test.
Twelve women underwent laparoscopic myomectomy within 169±16 minutes (mean±SEM) of preoperative UAE. Fourteen control patients underwent laparoscopic myomectomy alone. The UAE group had a greater mean preoperative clinical uterine size (19.7 versus 12.4 weeks, P<0.001) and a greater mean myoma specimen weight measured postoperatively (595.3 versus 153.6 grams, P<0.05). There were no significant differences in operative time or blood loss, and there were no intra-operative complications.
UAE performed immediately before laparoscopic myomectomy facilitated minimally invasive surgery for larger uteri and larger uterine myomas, with no differences in operative time or blood loss.
PMCID: PMC4034570  PMID: 22098788
Laparoscopy; Uterine artery embolization; Uterine leiomyomata
10.  Interannual variability in the timing of New England shellfish toxicity and relationships to environmental forcing 
Routine monitoring along the coast of the Gulf of Maine (GoM) reveals shellfish toxicity nearly every summer, but at varying times, locations, and magnitudes. The responsible toxin is known to be produced by the dinoflagellate Alexandrium fundyense, yet there is little apparent association between Alexandrium abundance and shellfish toxicity. One possibility is that toxic cells are persistent in offshore areas and variability in shellfish toxicity is caused not by changes in overall abundance, but rather by variability in transport processes. Measurements of offshore Alexandrium biomass are scarce, so we bypass cell abundance as an explanatory variable and focus instead on the relations between shellfish toxicity and concurrent metrics of GoM meteorology, hydrology, and oceanography. While this yields over two decades (1985–2005) of data representing a variety of interannual conditions, the toxicity data are gappy in spatial and temporal coverage. We address this through a combination of parametric curve fitting and hierarchical cluster analysis to reveal eight archetypical modes of seasonal toxicity timing. Groups of locations are then formed that have similar interannual patterns in these archetypes. Finally, the interannual patterns within each group are related to available environmental metrics using classification trees. Results indicate that a weak cross-shore sea surface temperature (SST) gradient in the summer is the strongest correlate of shellfish toxicity, likely by signifying a hydrological connection between offshore Alexandrium populations and near-shore shellfish beds. High cumulative downwelling wind strength early in the season is revealed as a precursor consistent with this mechanism. Although previous studies suggest that alongshore transport is important in moving Alexandrium from the eastern to western GoM, alongshore SST gradient is not an important correlate of toxicity in our study. We conclude by discussing the implications of our results for designing efficient and effective shellfish monitoring programs along the GoM coast.
PMCID: PMC3586711  PMID: 23391892
harmful algal blooms; red tides; paralytic shellfish poisoning; cluster analysis; CART modeling; satellite remote sensing
11.  Expanded Human Blood-Derived γδT Cells Display Potent Antigen-Presentation Functions 
Cell-based immunotherapy strategies target tumors directly (via cytolytic effector cells) or aim at mobilizing endogenous anti-tumor immunity. The latter approach includes dendritic cells (DC) most frequently in the form of in vitro cultured peripheral blood monocytes-derived DC. Human blood γδT cells are selective for a single class of non-peptide agonists (“phosphoantigens”) and develop into potent antigen-presenting cells (APC), termed γδT-APC within 1–3 days of in vitro culture. Availability of large numbers of γδT-APC would be advantageous for use as a novel cellular vaccine. We here report optimal γδT cell expansion (>107 cells/ml blood) when peripheral blood mononuclear cells (PBMC) from healthy individuals and melanoma patients were stimulated with zoledronate and then cultured for 14 days in the presence of IL-2 and IL-15, yielding γδT cell cultures of variable purity (77 ± 21 and 56 ± 26%, respectively). They resembled effector memory αβT (TEM) cells and retained full functionality as assessed by in vitro tumor cell killing as well as secretion of pro-inflammatory cytokines (IFNγ, TNFα) and cell proliferation in response to stimulation with phosphoantigens. Importantly, day 14 γδT cells expressed numerous APC-related cell surface markers and, in agreement, displayed potent in vitro APC functions. Day 14 γδT cells from PBMC of patients with cancer were equally effective as their counterparts derived from blood of healthy individuals and triggered potent CD8+ αβT cell responses following processing and cross-presentation of simple (influenza M1) and complex (tuberculin purified protein derivative) protein antigens. Of note, and in clear contrast to peripheral blood γδT cells, the ability of day 14 γδT cells to trigger antigen-specific αβT cell responses did not depend on re-stimulation. We conclude that day 14 γδT cell cultures provide a convenient source of autologous APC for use in immunotherapy of patients with various cancers.
PMCID: PMC4107971  PMID: 25101086
γδT cells; antigen-presentation; vaccine; αβT cells
12.  Impact of grazing intensity on seasonal variations in soil organic carbon and soil CO2 efflux in two semiarid grasslands in southern Botswana 
Biological soil crusts (BSCs) are an important source of organic carbon, and affect a range of ecosystem functions in arid and semiarid environments. Yet the impact of grazing disturbance on crust properties and soil CO2 efflux remain poorly studied, particularly in African ecosystems. The effects of burial under wind-blown sand, disaggregation and removal of BSCs on seasonal variations in soil CO2 efflux, soil organic carbon, chlorophyll a and scytonemin were investigated at two sites in the Kalahari of southern Botswana. Field experiments were employed to isolate CO2 efflux originating from BSCs in order to estimate the C exchange within the crust. Organic carbon was not evenly distributed through the soil profile but concentrated in the BSC. Soil CO2 efflux was higher in Kalahari Sand than in calcrete soils, but rates varied significantly with seasonal changes in moisture and temperature. BSCs at both sites were a small net sink of C to the soil. Soil CO2 efflux was significantly higher in sand soils where the BSC was removed, and on calcrete where the BSC was buried under sand. The BSC removal and burial under sand also significantly reduced chlorophyll a, organic carbon and scytonemin. Disaggregation of the soil crust, however, led to increases in chlorophyll a and organic carbon. The data confirm the importance of BSCs for C cycling in drylands and indicate intensive grazing, which destroys BSCs through trampling and burial, will adversely affect C sequestration and storage. Managed grazing, where soil surfaces are only lightly disturbed, would help maintain a positive carbon balance in African drylands.
PMCID: PMC3479694  PMID: 23045706
biological soil crusts; soil CO2 efflux; grazing; soil organic carbon
13.  A Quality Assurance Method that Utilizes 3D Dosimetry and Facilitates Clinical Interpretation 
International journal of radiation oncology, biology, physics  2012;84(2):10.1016/j.ijrobp.2011.12.015.
To demonstrate a new three-dimensional (3D) quality assurance (QA) method that provides comprehensive dosimetry verification and facilitates evaluation of the clinical significance of QA data acquired in a phantom. Also to apply the method to investigate the dosimetric efficacy of base-of-skull (BOS) intensity-modulated radiotherapy (IMRT) treatment.
Methods and Materials
Two types of IMRT QA verification plans were created for 6 patients who received BOS IMRT. The first plan enabled conventional 2D planar IMRT QA using the Varian portal dosimetry system. The second plan enabled 3D verification using an anthropomorphic head phantom. In the latter, the 3D dose distribution was measured using the DLOS/Presage dosimetry system (DLOS = Duke Large-field-of-view Optical-CT System, Presage Heuris Pharma, Skillman, NJ), which yielded isotropic 2-mm data throughout the treated volume. In a novel step, measured 3D dose distributions were transformed back to the patient’s CT to enable calculation of dose–volume histograms (DVH) and dose overlays. Measured and planned patient DVHs were compared to investigate clinical significance.
Close agreement between measured and calculated dose distributions was observed for all 6 cases. For gamma criteria of 3%, 2 mm, the mean passing rate for portal dosimetry was 96.8% (range, 92.0%–98.9%), compared to 94.9% (range, 90.1%–98.9%) for 3D. There was no clear correlation between 2D and 3D passing rates. Planned and measured dose distributions were evaluated on the patient’s anatomy, using DVH and dose overlays. Minor deviations were detected, and the clinical significance of these are presented and discussed.
Two advantages accrue to the methods presented here. First, treatment accuracy is evaluated throughout the whole treated volume, yielding comprehensive verification. Second, the clinical significance of any deviations can be assessed through the generation of DVH curves and dose overlays on the patient’s anatomy. The latter step represents an important development that advances the clinical relevance of complex treatment QA.
PMCID: PMC3832997  PMID: 22361085
3D dosimetry; IMRT; Quality assurance; Treatment verification
14.  Functional mechanotransduction is required for cisplatin-induced hair cell death in the zebrafish lateral line 
Cisplatin, one of the most commonly used anti-cancer drugs, is known to cause inner ear hair cell damage and hearing loss. Despite much investigation into mechanisms of cisplatin-induced hair cell death, little is known about the mechanism whereby cisplatin is selectively toxic to hair cells. Using hair cells of the zebrafish lateral line, we found that chemical inhibition of mechanotransduction with quinine and EGTA protected against cisplatin-induced hair cell death. Furthermore, we found that the zebrafish mutants mariner (myo7aa) and sputnik (cad23) that lack functional mechanotransduction were resistant to cisplatin-induced hair cell death. Using a fluorescent analogue of cisplatin, we found that chemical or genetic inhibition of mechanotransduction prevented its uptake. These findings demonstrate that cisplatin-induced hair cell death is dependent on functional mechanotransduction in the zebrafish lateral line.
PMCID: PMC3666553  PMID: 23467357
15.  Purkinje cell dysfunction and delayed death in plasma membrane calcium ATPase 2-heterozygous mice 
Purkinje cell (PC) dysfunction or death have been implicated in a number of disorders including ataxia, autism and multiple sclerosis. Plasma membrane calcium ATPase 2 (PMCA2), an important calcium (Ca2+) extrusion pump that interacts with synaptic signaling complexes, is most abundantly expressed in PCs compared to other neurons. Using the PMCA2 heterozygous mouse as a model, we investigated whether a reduction in PMCA2 levels affects PC function. We focused on Ca2+ signaling and the expression of glutamate receptors which play a key role in PC function including synaptic plasticity. We found that the amplitude of depolarization and 2-amino-3-(5-methyl-3-oxo-1,2-oxazol-4-yl)propanoic acid receptor (AMPAR)-mediated Ca2+ transients is significantly higher in cultured PMCA2+/− PCs than in PMCA2+/+ PCs. This is due to increased Ca2+ influx, since P/Q type voltage-gated Ca2+ channel (VGCC) expression was more pronounced in PCs and cerebella of PMCA2+/− mice and VGCC blockade prevented the elevation in amplitude. Neuronal nitric oxide synthase (nNOS) activity was higher in PMCA2+/−cerebella and inhibition of nNOS or the soluble guanylate cyclase (sGC)-cyclic guanosine monophosphate (cGMP) pathway, which mediates nitric oxide (NO) signaling, reduced the amplitude of Ca2+ transients in PMCA2+/− PCs, in vitro. In addition, there was an age-dependent decrease in metabotropic glutamate receptor 1 (mGluR1) and AMPA receptor subunit GluR2/3 transcript and protein levels at 8 weeks of age. These changes were followed by PC loss in the 20-week-old PMCA2+/− mice. Our studies highlight the importance of PMCA2 in Ca2+ signaling, glutamate receptor expression and survival of Purkinje cells.
PMCID: PMC3436994  PMID: 22789621
Ataxia; autism; neurodegeneration; glutamate; AMPA; calcium channel; ion pump; nitric oxide
16.  Calcium-Dependent Physiologic and Pathologic Stimulus-Metabolic Response Coupling in Hepatocytes 
Cell Calcium  2012;52(1):93-102.
A recurrent paradigm in calcium signaling is the coordination of the target response of the calcium signal with activation of metabolic energy production to support that response. This occurs in many tissues, including cardiac and skeletal muscle where contractile activity and ATP production are coordinately regulated by the frequency and amplitude of calcium transients, endocrine and exocrine cells that use calcium to drive the secretory process, and hepatocytes where the downstream targets of calcium include both catabolic and anabolic processes. The primary mechanism by which calcium enhances the capacity for energy production is through calcium-dependent stimulation of mitochondrial oxidative metabolism, achieved by increasing NADH production and respiratory chain flux. Although this enhances energy supply, it also has the potential for deleterious consequences resulting from increased generation of reactive oxygen species (ROS). The negative consequences of calcium-dependent mitochondrial activation can be ameliorated when the underlying cytosolic calcium signals occur as brief calcium spikes or oscillations, with signal strength encoded through the spike frequency (frequency modulation). Frequency modulation increases signal fidelity, and reduces pathological effects of calcium, including excess mitochondrial ROS production and apoptotic or necrotic outcomes. The present article reviews these issues using data obtained in hepatocytes under physiologic and pathologic conditions.
PMCID: PMC3391328  PMID: 22564906
17.  Homophily and Contagion Are Generically Confounded in Observational Social Network Studies 
Sociological methods & research  2011;40(2):211-239.
The authors consider processes on social networks that can potentially involve three factors: homophily, or the formation of social ties due to matching individual traits; social contagion, also known as social influence; and the causal effect of an individual’s covariates on his or her behavior or other measurable responses. The authors show that generically, all of these are confounded with each other. Distinguishing them from one another requires strong assumptions on the parametrization of the social process or on the adequacy of the covariates used (or both). In particular the authors demonstrate, with simple examples, that asymmetries in regression coefficients cannot identify causal effects and that very simple models of imitation (a form of social contagion) can produce substantial correlations between an individual’s enduring traits and his or her choices, even when there is no intrinsic affinity between them. The authors also suggest some possible constructive responses to these results.
PMCID: PMC3328971  PMID: 22523436
contagion; social influence; homophily; causal inference; network confounding; neutral models
18.  Use of thermal melt curves to assess the quality of enzyme preparations1 
Analytical Biochemistry  2009;399(2):268-275.
This study sought to determine whether the quality of enzyme preparations can be determined from their melting curves, which may easily be obtained using a fluorescent probe and a standard RT-PCR machine. Thermal melt data on 31 recombinant enzymes from Plasmodium parasites were acquired by incrementally heating them to 90 °C and measuring unfolding with a fluorescent dye; activity assays specific to each enzyme were also performed. Four of the enzymes were denatured to varying degrees with heat and SDS prior to the thermal melt and activity assays. In general, melting curve quality correlated with enzyme activity; enzymes with high-quality curves were found almost uniformly to be active, while those with lower-quality curves were more varied in their catalytic performance. Inspection of melting curves of bovine xanthine oxidase and Entamoeba histolytica cysteine protease 1 allowed active stocks to be distinguished from inactive stocks, implying that a relationship between melting curve quality and activity persists over a wide range of experimental conditions and species. Our data suggest that melting curves can help to distinguish properly folded proteins from denatured ones and therefore may be useful in selecting stocks for further study and in optimizing purification procedures for specific proteins.
PMCID: PMC3270315  PMID: 20018159
thermal melting; malaria; protein denaturation
19.  M2 macrophages exhibit higher sensitivity to oxLDL-induced lipotoxicity than other monocyte/macrophage subtypes 
In obesity, phenotypic switches occur in macrophage populations such that the predominantly M2-polarised anti-inflammatory state seen in lean individuals changes to a predominantly M1-polarised pro-inflammatory state in those who are obese. However, the mechanisms by which these phenotypic shifts occur have not yet been fully elucidated.
The effects of oxLDL (1-40 μg/ml; 24 h) on several parameters relevant to the Unfolded Protein Response (UPR)-mediated lipotoxic effects of oxLDL (disruption of ER Ca2+ handling; activation of the UPR transcription factor XBP-1; upregulation of the UPR target genes BiP and CHOP; apoptosis; cell viability) were investigated in human primary monocyte-derived macrophages, and also in monocyte-macrophages derived from the THP-1 monocytic cell line. A consistent pattern was observed: M2-polarised macrophages were more sensitive to the lipotoxic effects of oxLDL than either non-polarised macrophages or non-differentiated monocytic cells. Specifically, M2-polarised macrophages were the only cell type to undergo significantly increased apoptosis (Primary cells: 1.23 ± 0.01 basal; THP-1-derived: 1.97 ± 0.12 basal; P < 0.05 in both cases) and decreased cell viability (Primary cells: 0.79 ± 0.04 basal; THP-1-derived: 0.67 ± 0.02 basal; P < 0.05 in both cases) when exposed to oxLDL levels similar to those seen in overweight individuals (ie. 1 μg/ml).
We propose that the enhanced susceptibility of M2-polarised macrophages to lipotoxicity seen in the present in vitro study could, over time, contribute to the phenotypic shift seen in obese individuals in vivo. This is because a higher degree of oxLDL-induced lipotoxic cell death within M2 macrophages could contribute to a decrease in numbers of M2 cells, and thus a relative increase in proportion of non-M2 cells, within macrophage populations. Given the pro-inflammatory characteristics of a predominantly M1-polarised state, the data presented here may constitute a useful contribution to our understanding of the origin of the pro-inflammatory nature of obesity, and of the pathogenesis of obesity-associated inflammatory disorders such as Type 2 Diabetes and atherosclerosis.
PMCID: PMC3281809  PMID: 22146099
alternative M2 monocyte/macrophage polarisation; UPR; oxLDL; lipotoxicity
20.  A method to correct for stray light in telecentric optical-CT imaging of radiochromic dosimeters 
Physics in medicine and biology  2011;56(14):4433-4451.
Radiochromic plastic and gel materials have recently emerged which can yield 3D dose information over clinical volumes in high resolution. These dosimeters can provide a much more comprehensive verification of complex radiation therapy treatments than can be achieved by conventional planar and point dosimeters. To achieve full clinical potential, these dosimeters require a fast and accurate read-out technology. Broad-beam optical-computed tomography (optical-CT) systems have shown promise, but can be sensitive to stray light artifacts originating in the imaging chain. In this work we present and evaluate a method to correct for stray light artifacts by deconvolving a measured, spatially invariant, point spread function (PSF). The correction was developed for the DLOS (Duke large field-of-view optical-CT scanner) in conjunction with radiochromic PRESAGE® dosimeters. The PSF was constructed from a series of acquisitions of projection images of various sized apertures placed in the optical imaging chain. Images were acquired with a range of exposure times, and for a range of aperture sizes (0.2–11 mm). The PSF is investigated under a variety of conditions, and found to be robust and spatially invariant, key factors enabling the viability of the deconvolution approach. The spatial invariance and robustness of the PSF are facilitated by telecentric imaging, which produces a collimated light beam and removes stray light originating upstream of the imaging lens. The telecentric capability of the DLOS therefore represents a significant advantage, both in keeping stray light levels to a minimum and enabling viability of an accurate PSF deconvolution method to correct for the residual. The performance of the correction method was evaluated on projection images containing known optical-density variations, and also on known 3D dose distributions. The method is shown to accurately account for stray light on small field dosimetry with corrections up to 3% in magnitude shown here although corrections of >10% have been observed in extreme cases. The dominant source of stray light was found to be within the imaging lens. Correcting for stray light extended the dynamic range of the system from ~30 to ~60 dB. The correction should be used when measurements need to be accurate within 3%.
PMCID: PMC3227692  PMID: 21719946
21.  A method to correct for spectral artifacts in optical-CT dosimetry 
Physics in medicine and biology  2011;56(11):3403-3416.
The recent emergence of radiochromic dosimeters with low inherent light-scattering presents the possibility of fast 3D dosimetry using broad-beam optical computed tomography (optical-CT). Current broad beam scanners typically employ either a single or a planar array of light-emitting diodes (LED) for the light source. The spectrum of light from LED sources is polychromatic and this, in combination with the non-uniform spectral absorption of the dosimeter, can introduce spectral artifacts arising from preferential absorption of photons at the peak absorption wavelengths in the dosimeter. Spectral artifacts can lead to large errors in the reconstructed attenuation coefficients, and hence dose measurement. This work presents an analytic method for correcting for spectral artifacts which can be applied if the spectral characteristics of the light source, absorbing dosimeter, and imaging detector are known or can be measured. The method is implemented here for a PRESAGE® dosimeter scanned with the DLOS telecentric scanner (Duke Large field-of-view Optical-CT Scanner). Emission and absorption profiles were measured with a commercial spectrometer and spectrophotometer, respectively. Simulations are presented that show spectral changes can introduce errors of 8% for moderately attenuating samples where spectral artifacts are less pronounced. The correction is evaluated by application to a 16 cm diameter PRESAGE® cylindrical dosimeter irradiated along the axis with two partially overlapping 6 × 6 cm fields of different doses. The resulting stepped dose distribution facilitates evaluation of the correction as each step had different spectral contributions. The spectral artifact correction was found to accurately correct the reconstructed coefficients to within ~1.5%, improved from ~7.5%, for normalized dose distributions. In conclusion, for situations where spectral artifacts cannot be removed by physical filters, the method shown here is an effective correction. Physical filters may be less viable if they introduce strong sensitivity to Schlieren bands in the dosimeters.
PMCID: PMC3227693  PMID: 21572184
22.  A comprehensive method for optical-emission computed tomography 
Physics in medicine and biology  2010;55(14):3947-3957.
Optical-computed tomography (CT) and optical-emission computed tomography (ECT) are recent techniques with potential for high-resolution multi-faceted 3D imaging of the structure and function in unsectioned tissue samples up to 1–4 cc. Quantitative imaging of 3D fluorophore distribution (e.g. GFP) using optical-ECT is challenging due to attenuation present within the sample. Uncorrected reconstructed images appear hotter near the edges than at the center. A similar effect is seen in SPECT/PET imaging, although an important difference is attenuation occurs for both emission and excitation photons. This work presents a way to implement not only the emission attenuation correction utilized in SPECT, but also excitation attenuation correction and source strength modeling which are unique to optical-ECT. The performance of the correction methods was investigated by the use of a cylindrical gelatin phantom whose central region was filled with a known distribution of attenuation and fluorophores. Uncorrected and corrected reconstructions were compared to a sectioned slice of the phantom imaged using a fluorescent dissecting microscope. Significant attenuation artifacts were observed in uncorrected images and appeared up to 80% less intense in the central regions due to attenuation and an assumed uniform light source. The corrected reconstruction showed agreement throughout the verification image with only slight variations (~5%). Final experiments demonstrate the correction in tissue as applied to a tumor with constitutive RFP.
PMCID: PMC3227695  PMID: 20577042
23.  The role of leukocyte-stromal interactions in chronic inflammatory joint disease 
Rheumatoid arthritis (RA) is a debilitating, chronic, persistent inflammatory disease that is characterised by painful and swollen joints. The aetiology of RA is unknown, however whereas past research has concentrated on the role of immune or inflammatory infiltrating cells in inflammation, it is becoming clear that stromal cells play a critical part in regulating the quality and duration of an inflammatory response. In this review we assess the role of fibroblasts within the inflamed synovium in modulating immune responses; in particular we examine the role of stromal cells in the switch from resolving to persistent inflammation as is found in the rheumatoid synovium.
PMCID: PMC3122102  PMID: 15681242
Rheumatoid arthritis; Cytokines; Chemokines; Synovitis; Inflammation
24.  Differential Survival of Leukocyte Subsets Mediated by Synovial, Bone Marrow, and Skin Fibroblasts 
Arthritis and rheumatism  2006;54(7):2096-2108.
Synovial fibroblasts share a number of phenotype markers with fibroblasts derived from bone marrow. In this study we investigated the role of matched fibroblasts obtained from 3 different sources (bone marrow, synovium, and skin) to test the hypothesis that synovial fibroblasts share similarities with bone marrow–derived fibroblasts in terms of their ability to support survival of T cells and neutrophils.
Matched synovial, bone marrow, and skin fibroblasts were established from 8 different patients with rheumatoid arthritis who were undergoing knee or hip surgery. Resting or activated fibroblasts were cocultured with either CD4 T cells or neutrophils, and the degree of leukocyte survival, apoptosis, and proliferation were measured.
Fibroblasts derived from all 3 sites supported increased survival of CD4 T cells, mediated principally by interferon-β. However, synovial and bone marrow fibroblasts shared an enhanced site-specific ability to maintain CD4 T cell survival in the absence of proliferation, an effect that was independent of fibroblast activation or proliferation but required direct T cell–fibroblast cell contact. In contrast, fibroblast-mediated neutrophil survival was less efficient, being independent of the site of origin of the fibroblast but dependent on prior fibroblast activation, and mediated solely by soluble factors, principally granulocyte–macrophage colony-stimulating factor.
These results suggest an important functional role for fibroblasts in the differential accumulation of leukocyte subsets in a variety of tissue microenvironments. The findings also provide a potential explanation for site-specific differences in the pattern of T cell and neutrophil accumulation observed in chronic inflammatory diseases.
PMCID: PMC3119431  PMID: 16802344
25.  Galectin 3 Induces a Distinctive Pattern of Cytokine and Chemokine Production in Rheumatoid Synovial Fibroblasts via Selective Signaling Pathways 
Arthritis and rheumatism  2009;60(6):1604-1614.
High expression of galectin 3 at sites of joint destruction in rheumatoid arthritis (RA) suggests that galectin 3 plays a role in RA pathogenesis. Previous studies have demonstrated the effects of galectins on immune cells, such as lymphocytes and macrophages. This study was undertaken to investigate the hypothesis that galectin 3 induces proinflammatory effects in RA by modulating the pattern of cytokine and chemokine production in synovial fibroblasts.
Matched samples of RA synovial and skin fibroblasts were pretreated with galectin 3 or tumor necrosis factor α (TNFα), and the levels of a panel of cytokines, chemokines, and matrix metalloproteinases (MMPs) were determined using enzyme-linked immunosorbent assays and multiplex assays. Specific inhibitors were used to dissect signaling pathways, which were confirmed by Western blotting and NF-κB activation assay.
Galectin 3 induced secretion of interleukin-6 (IL-6), granulocyte–macrophage colony-stimulating factor, CXCL8, and MMP-3 in both synovial and skin fibroblasts. By contrast, galectin 3–induced secretion of TNFα, CCL2, CCL3, and CCL5 was significantly greater in synovial fibroblasts than in skin fibroblasts. TNFα blockade ruled out autocrine TNFα-stimulated induction of chemokines. The MAPKs p38, JNK, and ERK were necessary for IL-6 production, but phosphatidylinositol 3-kinase (PI 3-kinase) was required for selective CCL5 induction. NF-κB activation was required for production of both IL-6 and CCL5.
Our findings indicate that galectin 3 promotes proinflammatory cytokine secretion by tissue fibroblasts. However, galectin 3 induces the production of mononuclear cell–recruiting chemokines uniquely from synovial fibroblasts, but not matched skin fibroblasts, via a PI 3-kinase signaling pathway. These data provide further evidence of the role of synovial fibroblasts in regulating the pattern and persistence of the inflammatory infiltrate in RA and suggest a new and important functional consequence of the observed high expression of galectin 3 in the rheumatoid synovium.
PMCID: PMC3116228  PMID: 19479862

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