Peter J S Smith

Università degli Studi G. d'Annunzio Chieti e Pescara, Chieta, Abruzzo, Italy

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Publications (97)

  • Ishna N. Mistry · Peter J.S. Smith · David I. Wilson · Ali Tavassoli
    [Show abstract] [Hide abstract] ABSTRACT: HIF-1 is the master regulator of cellular hypoxia response; the oxygen sensitive HIF-1α subunit transactivates its own expression in hypoxia by binding to a hypoxia response element (HRE) in the promoter of the HIF-1α gene. This transactivation loop significantly contributes to the build up of HIF-1α at the onset of hypoxia, with the binding of HIF-1 to the HIF-1α promoter being dependent on the epigenetic status of a CpG dinucleotide in the upstream HRE. Given the central role played by HIF-1 in tissue development, we sought to probe the epigenetic status of the HIF-1α HRE and that of its downstream target EPO in embryonic tissue. Our data shows that the CpG dinucleotide in HIF-1α HRE is unmethylated in several embryonic tissue samples, suggesting that transactivation of HIF-1α plays a significant role in HIF-1 mediated hypoxia response during development.
    Article · Jun 2015 · Molecular BioSystems
  • [Show abstract] [Hide abstract] ABSTRACT: Inefficiency of oxidative phosphorylation can result from futile leak conductance through the inner mitochondrial membrane. Stress or injury may exacerbate this leak conductance, putting cells, and particularly neurons, at risk of dysfunction and even death when energy demand exceeds cellular energy production. Using a novel method, we have recently described an ion conductance consistent with mitochondrial permeability transition pore (mPTP) within the c-subunit of the ATP synthase. Excitotoxicity, ROS producing stimuli or elevated mitochondrial matrix calcium open the channel which is inhibited by cyclosporine A (CsA) and ATP/ADP. Here we show that ATP, and the neuroprotective drug dexpramipexole (DEX; KNS-760704; ((6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine) inhibited an ion conductance consistent with this c-subunit channel (mPTP) in brain-derived submitochondrial vesicles (SMVs) enriched for F1FO ATP synthase (complex V). Treatment of SMVs with urea denatured extramembrane components of complex V, eliminated DEX but not ATP-mediated current inhibition and reduced binding of (14)C-DEX. Direct effects of DEX on the synthesis and hydrolysis of ATP by complex V suggest that interaction of the compound with its target results in functional conformational changes in the enzyme complex. (14)C-DEX bound specifically to purified recombinant b and OSCP subunits of the mitochondrial F1FO ATP synthase. Previous data indicate that DEX, increased the efficiency of energy production in cells, including neurons. Taken together, these studies suggest that modulation of a complex V-associated inner mitochondrial membrane current is metabolically important and may represent an avenue for the development of new therapeutics for neurodegenerative disorders.
    Article · Oct 2014 · Molecular pharmacology
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    [Show abstract] [Hide abstract] ABSTRACT: We report the Laser Induced Forward Transfer (LIFT) of antibodies from a liquid donor film onto paper receivers for application as point-of-care diagnostic sensors. To minimise the loss of functionality of the active biomolecules during transfer, a dynamic release layer was employed to shield the biomaterial from direct exposure to the pulsed laser source. Cellulose paper was chosen as the ideal receiver because of its inherent bio-compatibility, liquid transport properties, wide availability and low cost, all of which make it an efficient and suitable platform for point-of-care diagnostic sensors. Both enzyme-tagged and untagged IgG antibodies were LIFT-printed and their functionality was confirmed via a colorimetric enzyme-linked immunosorbent assay. Localisation of the printed antibodies was exhibited, which can allow the creation of complex 2-d patterns such as QR codes or letters for use in a final working device. Finally, a calibration curve was determined that related the intensity of the colour obtained to the concentration of active antibodies to enable quantitative assessment of the device performance. The motivation for this work was to implement a laser-based procedure for manufacturing low-cost, point-of-care diagnostic devices on paper.
    Full-text Article · May 2014 · Biomicrofluidics
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    [Show abstract] [Hide abstract] ABSTRACT: The behavior and genetics of serous epithelial ovarian cancer (EOC) metastasis, the form of the disease lethal to patients, is poorly understood. The unique properties of metastases are critical to understand to improve treatments of the disease that remains in patients after debulking surgery. We sought to identify the genetic and phenotypic landscape of metastatic progression of EOC to understand how metastases compare to primary tumors. DNA copy number and mRNA expression differences between matched primary human tumors and omental metastases, collected at the same time during debulking surgery before chemotherapy, were measured using microarrays. qPCR and immunohistochemistry validated findings. Pathway analysis of mRNA expression revealed metastatic cancer cells are more proliferative and less apoptotic than primary tumors, perhaps explaining the aggressive nature of these lesions. Most cases had copy number aberrations (CNAs) that differed between primary and metastatic tumors, but we did not detect CNAs that are recurrent across cases. A six gene expression signature distinguishes primary from metastatic tumors and predicts overall survival in independent datasets. The genetic differences between primary and metastatic tumors, yet common expression changes, suggest that the major clone in metastases is not the same as in primary tumors, but the cancer cells adapt to the omentum similarly. Together, these data highlight how ovarian tumors develop into a distinct, more aggressive metastatic state that should be considered for therapy development.
    Full-text Article · Apr 2014 · PLoS ONE
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    Benjamin A S Van Mooy · Laura R Hmelo · Helen F Fredricks · [...] · Peter J S Smith
    [Show abstract] [Hide abstract] ABSTRACT: The accumulation of microbial biofilms on ships' hulls negatively affects ship performance and efficiency while also playing a role in the establishment of even more detrimental hard-fouling communities. However, there is little quantitative information on how the accumulation rate of microbial biofilms is impacted by the balance of the rates of cell settlement, in situ production (ie growth), dispersal to surrounding waters and mortality induced by grazers. These rates were quantified on test panels coated with copper-based antifouling (AF) or polymer-based fouling-release (FR) coatings by using phospholipids as molecular proxies for microbial biomass. The results confirmed the accepted modes of efficacy of these two types of coatings. In a more extensive set of experiments with only the FR coatings, it was found that seasonally averaged cellular production rates were 1.5 ± 0.5 times greater than settlement and the dispersal rates were 2.7 ± 0.8 greater than grazing. The results of this study quantitatively describe the dynamic balance of processes leading to the accumulation of microbial biofilm on coatings designed for ships' hulls.
    Full-text Article · Jan 2014 · Biofouling
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    [Show abstract] [Hide abstract] ABSTRACT: The pace of development in the field of advanced microscopy is truly breath-taking, and is leading to major breakthroughs in our understanding of molecular machines and cell function. This special issue of Journal of Optics draws attention to a number of interesting approaches, ranging from fluorescence and imaging of unlabelled cells, to computational methods, all of which are describing the ever increasing detail of the dynamic behaviour of molecules in the living cell. This is a field which traditionally, and currently, demonstrates a marvellous interplay between the disciplines of physics, chemistry and biology, where apparent boundaries to resolution dissolve and living cells are viewed in ever more clarity. It is fertile ground for those interested in optics and non-conventional imaging to contribute high-impact outputs in the fields of cell biology and biomedicine. The series of articles presented here has been selected to demonstrate this interdisciplinarity and to encourage all those with a background in the physical sciences to 'dip their toes' into the exciting and dynamic discoveries surrounding cell function. Although single molecule super-resolution microscopy is commercially available, specimen preparation and interpretation of single molecule data remain a major challenge for scientists wanting to adopt the techniques. The paper by Allen and Davidson [1] provides a much needed detailed introduction to the practical aspects of stochastic optical reconstruction microscopy, including sample preparation, image acquisition and image analysis, as well as a brief description of the different variants of single molecule localization microscopy. Since super-resolution microscopy is no longer restricted to three-dimensional imaging of fixed samples, the review by Fiolka [2] is a timely introduction to techniques that have been successfully applied to four-dimensional live cell super-resolution microscopy. The combination of multiple high-resolution techniques, such as the combination of light sheet and structured illumination microscopy (SIM), which efficiently utilize photon budget and avoid illuminating regions of the specimen not currently being imaged, hold the greatest promise for future biological applications. Therefore, the combined setup for SIM and single molecule localization microscopy (SMLM) described by Rossberger et al [3] will be very helpful and stimulating to advanced microscopists in further modifying their setups. The SIM image helps in identifying artefacts in SMLM reconstruction, e.g. when two active fluorophores are close together and get rejected as 'out-of-focus'. This combined setup is another way to facilitate imaging live samples. The article by Thomas et al [4] presents another advance for biological super-resolution imaging with a new approach to reconstruct optically sectioned images using structured illumination. The method produces images with higher spatial resolution and greater signal to noise compared to existing approaches. This algorithm demonstrates great promise for reconstructing biological images where the signal intensities are inherently lower. Shevchuk et al [5] present a non-optic near field approach to imaging with a review of scanning ion-conductance microscopy. This is a powerful alternative approach for examining the surface dynamics of living cells including exo and endocytosis, unlabelled, and at the level of the single event. Here they present the first data on combining this approach with fluorescence confocal microscopy—adding that extra dimension. Different approaches to label-free live cell imaging are presented in the papers by Patel et al [6], Mehta and Oldenbourg [7], as well as Rogers and Zheludev [8]. All three papers bring home the excitement of looking at live cell dynamics without reporters—Patel et al [6] review both the potential of coherent anti-Stokes Raman scattering and biological applications, where specific biomolecules are detected on the basis of their biophysical properties. Polarized light microscopy as presented by Mehta and Oldenbourg [7], describe a novel implementation of this technology to detect dichroism, and demonstrate beautifully its use in imaging unlabelled microtubules, mitochondria and lipid droplets. Sub-wavelength light focusing provides another avenue to super-resolution, and this is presented by Rogers and Zheludev [8]. Speculating on further improvements, these authors expect a resolution of 0.15λ. To date, the method has not been applied to low contrast, squishy and motile biotargets, but is included here for the clear potential to drive label-free imaging in new directions. A similar logic lies behind the inclusion of Parsons et al [9] where ultraviolet coherent diffractive imaging is further developed. These authors have demonstrated a shrink-wrap technique which reduces the integration time by a factor of 5, bringing closer the time when we have lab based imaging systems based on extreme ultraviolet and soft x-ray sources using sophisticated phase retrieval algorithms. Real biological specimens have spatially varying refractive indices that inevitably lead to aberrations and image distortions. Global refractive index matching of the embedding medium has been an historic solution, but unfortunately is not practical for live cell imaging. Adaptive optics appears an attractive solution and Simmonds and Booth [10] demonstrate the theoretical benefits of applying several adaptive optical elements, placed in different conjugate planes, to create a kind of 'inverse specimen' that unwarps phase distortions of the sample—but these have yet to be tested on real specimens. A difficulty in single molecule localization microscopy has been the determination of whether or not two molecules are colocalized. Kim et al [11] present a method for correcting bleed-through during multi-colour, single molecule localization microscopy. Such methods are welcome standards when trying to quantifiably interpret how close two molecules actually are. Rees et al [12] provide an invaluable overview of key image processing steps in localization microscopy. This paper is an excellent starting point for anyone implementing localization algorithms and the Matlab software provided will be invaluable; a strong paper on which to conclude our overview of the excellent articles brought together in this issue. One aspect brought home in several of these articles is the volume of data now being collected by high resolution live cell imaging. Data processing and image reconstruction will continue to be pressure points in the further development of instrumentation and analyses. We would hope that the series of papers presented here will motivate software engineers, optical physicists and biologists to contribute to the further development of this exciting field. References [1] Allen J R et al 2013 J. Opt. 15 094001 [2] Fiolka R et al 2013 J. Opt. 15 094002 [3] Rossberger S et al 2013 J. Opt. 15 094003 [4] Thomas B et al 2013 J. Opt. 15 094004 [5] Shevchuk A et al 2013 J. Opt. 15 094005 [6] Patel I et al 2013 J. Opt. 15 094006 [7] Mehta S B et al 2013 J. Opt. 15 094007 [8] Rogers E T F et al 2013 J. Opt. 15 094008 [9] Parsons A D et al 2013 J. Opt. 15 094009 [10] Simmonds R et al 2013 J. Opt. 15 094010 [11] Kim D et al 2013 J. Opt. 15 094011 [12] Rees E J et al 2013 J. Opt. 15 094012
    Full-text Article · Sep 2013 · Journal of Optics
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    Dataset: Figure S6
    [Show abstract] [Hide abstract] ABSTRACT: Pictures of the H&E stained cancer cells before and after laser capture microdissection (LCM). Pockets of cancer cells were selected for removal and analysis. (TIF)
    Full-text Dataset · Mar 2013
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    Dataset: Figure S2
    [Show abstract] [Hide abstract] ABSTRACT: Bar graph summary of miRNA Taqman expression data shown in Figure 1A highlighting the miRNA expression changes in each tumor. (TIF)
    Full-text Dataset · Mar 2013
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    [Show abstract] [Hide abstract] ABSTRACT: Serous epithelial ovarian cancer (EOC) patients often succumb to aggressive metastatic disease, yet little is known about the behavior and genetics of ovarian cancer metastasis. Here, we aim to understand how omental metastases differ from primary tumors and how these differences may influence chemotherapy. We analyzed the miRNA expression profiles of primary EOC tumors and their respective omental metastases from 9 patients using miRNA Taqman qPCR arrays. We find 17 miRNAs with differential expression in omental lesions compared to primary tumors. miR-21, miR-150, and miR-146a have low expression in most primary tumors with significantly increased expression in omental lesions, with concomitant decreased expression of predicted mRNA targets based on mRNA expression. We find that miR-150 and miR-146a mediate spheroid size. Both miR-146a and miR-150 increase the number of residual surviving cells by 2-4 fold when challenged with lethal cisplatin concentrations. These observations suggest that at least two of the miRNAs, miR-146a and miR-150, up-regulated in omental lesions, stimulate survival and increase drug tolerance. Our observations suggest that cancer cells in omental tumors express key miRNAs differently than primary tumors, and that at least some of these microRNAs may be critical regulators of the emergence of drug resistant disease.
    Full-text Article · Mar 2013 · PLoS ONE
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    Dataset: Figure S1
    [Show abstract] [Hide abstract] ABSTRACT: Tumors are of ovarian origin and are serous epithelial as indicated from examination of H&E and cytokeratin staining. Representative H&E staining of two representative cases. CA125 and cytokeratin staining of one case is consistent with ovarian tumor origins. (TIF)
    Full-text Dataset · Mar 2013
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    Dataset: Figure S7
    [Show abstract] [Hide abstract] ABSTRACT: The 8 miRNAs expressed in both cell lines and LCM enriched cancer cells in tumors have increased expression in spheroids compared to monolayer cell culture. Taqman qPCR array card data of miRNA expression in monolayer and spheroids. Fold changes calculated by ΔCt method normalized to U6 snRNA. (TIF)
    Full-text Dataset · Mar 2013
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    Dataset: Figure S8
    [Show abstract] [Hide abstract] ABSTRACT: miR-146a expression after 24 hours after transfection with 50 nM pre-miR in SKOV-3 and OVCAR-8 cells using Taqman assays targeting only miR-146a. Fold changes calculated by ΔCt method normalized to U6 snRNA. Control are cells transfected with negative control pre-miR. Error bars are standard deviation from three independent experiments transfected in parallel with the functional assays. (TIF)
    Full-text Dataset · Mar 2013
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    Dataset: Figure S5
    [Show abstract] [Hide abstract] ABSTRACT: In situ hybridization of miR-21 in matched primary tumors and omental metastases. Cancer cells are stained red by Nuclear Red. (TIF)
    Full-text Dataset · Mar 2013
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    Dataset: Figure S4
    [Show abstract] [Hide abstract] ABSTRACT: miR-146a and miR-150 predicted mRNA targets are significantly down-regulated in omental lesions compared to primary tumors. A. Global distribution of the Pearson correlation coefficients between mRNAs and miRNAs in the primary and metastatic tumors. Red line indicates mRNA targets from the union of TargetScan and PITA predictions. Grey lines are randomly selected sets of transcripts of the same size permuted 1,000 times. P-values are calculated by counting the number of distributions with means lower than the target distribution to define the background. B. Genes with Pearson correlation coefficients<−0.3 in the tumors are significantly enriched for specific pathways and functions as determined by Ingenuity Pathway Analysis (IPA). P-values are multiple hypothesis corrected using Benjamini-Hochberg (3). Selected genes for each pathway are listed. (TIF)
    Full-text Dataset · Mar 2013
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    Dataset: Figure S3
    [Show abstract] [Hide abstract] ABSTRACT: Taqman assays targeting individual miRNAs and U6 snRNA are consistent with megaplex pooled Taqman assays from bulk tumor purified RNA. Indiv indicates assay perform with primers only for the designated miRNA. Megaplex is the bulk tumor fold change from the pooled 377 miRNA assay used for the screen shown in Figure 1. All fold changes are calculated using the ΔCt method relative to U6 snRNA. Data for case 1 and 3 are shown. (TIF)
    Full-text Dataset · Mar 2013
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    Dataset: Table S1
    [Show abstract] [Hide abstract] ABSTRACT: Summary of Patient Characteristics. (XLSX)
    Full-text Dataset · Mar 2013
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    [Show abstract] [Hide abstract] ABSTRACT: Cellular stress or injury can result in mitochondrial dysfunction, which has been linked to many chronic neurological disorders including amyotrophic lateral sclerosis (ALS) and Parkinson's disease (PD). Stressed and dysfunctional mitochondria exhibit an increase in large conductance mitochondrial membrane currents and a decrease in bioenergetic efficiency. Inefficient energy production puts cells, and particularly neurons, at risk of death when energy demands exceed cellular energy production. Here we show that the candidate ALS drug dexpramipexole (DEX; KNS-760704; ((6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine) and cyclosporine A (CSA) inhibited increases in ion conductance in whole rat brain-derived mitochondria induced by calcium or treatment with a proteasome inhibitor, although only CSA inhibited calcium-induced permeability transition in liver-derived mitochondria. In several cell lines, including cortical neurons in culture, DEX significantly decreased oxygen consumption while maintaining or increasing production of adenosine triphosphate (ATP). DEX also normalized the metabolic profile of injured cells and was protective against the cytotoxic effects of proteasome inhibition. These data indicate that DEX increases the efficiency of oxidative phosphorylation, possibly by inhibition of a CSA-sensitive mitochondrial conductance.
    Full-text Article · Jan 2012 · Brain research
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    [Show abstract] [Hide abstract] ABSTRACT: Pancreatic β-cells release insulin in response to elevation of glucose from basal (4-7mM) to stimulatory (8-16mM) levels. Metabolism of glucose by the β-cell results in the production of low levels of reactive oxygen intermediates (ROI), such as hydrogen peroxide (H(2)O(2)), a newly recognized coupling factor linking glucose metabolism to insulin secretion. However, high and toxic levels of H(2)O(2) inhibit insulin secretion. Menadione, which produces H(2)O(2) via redox cycling mechanism in a dose-dependent manner, was investigated for its effect on β-cell metabolism and insulin secretion in INS-1 832/13, a rat β-cell insulinoma cell line, and primary rodent islets. Menadione-dependent redox cycling and resulting H(2)O(2) production under stimulatory glucose exceeded several-fold those reached at basal glucose. This was paralleled by a differential effect of menadione (0.1-10μM) on insulin secretion, which was enhanced at basal, but inhibited at stimulatory glucose. Redox cycling of menadione and H(2)O(2) formation was dependent on glycolytically-derived NADH, as inhibition of glycolysis and application of non-glycogenic insulin secretagogues did not support redox cycling. In addition, activity of plasma membrane electron transport, a system dependent in part on glycolytically-derived NADH, was also inhibited by menadione. Menadione-dependent redox cycling was sensitive to the NQO1 inhibitor dicoumarol and the flavoprotein inhibitor diphenylene iodonium, suggesting a role for NQO1 and other oxidoreductases in this process. These data may explain the apparent dichotomy between the stimulatory and inhibitory effects of H(2)O(2) and menadione on insulin secretion.
    Full-text Article · Nov 2011 · Toxicology and Applied Pharmacology
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    Full-text Article · Nov 2011 · Nature Cell Biology
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    [Show abstract] [Hide abstract] ABSTRACT: Anti-apoptotic Bcl2 family proteins such as Bcl-x(L) protect cells from death by sequestering apoptotic molecules, but also contribute to normal neuronal function. We find in hippocampal neurons that Bcl-x(L) enhances the efficiency of energy metabolism. Our evidence indicates that Bcl-x(L)interacts directly with the β-subunit of the F(1)F(O) ATP synthase, decreasing an ion leak within the F(1)F(O) ATPase complex and thereby increasing net transport of H(+) by F(1)F(O) during F(1)F(O) ATPase activity. By patch clamping submitochondrial vesicles enriched in F(1)F(O) ATP synthase complexes, we find that, in the presence of ATP, pharmacological or genetic inhibition of Bcl-x(L) activity increases the membrane leak conductance. In addition, recombinant Bcl-x(L) protein directly increases the level of ATPase activity of purified synthase complexes, and inhibition of endogenous Bcl-x(L) decreases the level of F(1)F(O) enzymatic activity. Our findings indicate that increased mitochondrial efficiency contributes to the enhanced synaptic efficacy found in Bcl-x(L)-expressing neurons.
    Full-text Article · Sep 2011 · Nature Cell Biology

Publication Stats

2k Citations

Institutions

  • 2011
    • Università degli Studi G. d'Annunzio Chieti e Pescara
      • Department of Neuroscience & Imaging
      Chieta, Abruzzo, Italy
  • 2007
    • Boca Raton Regional Hospital
      Boca Raton, Florida, United States
    • Harvard University
      Cambridge, Massachusetts, United States
  • 2000-2007
    • Marine Biological Laboratory
      • BioCurrents Research Center
      New York City, NY, United States
    • Brown University
      Providence, Rhode Island, United States
    • Women & Infants Hospital
      Providence, Rhode Island, United States
  • 2003
    • Yale University
      New Haven, Connecticut, United States