Single-Cell Mass Spectrometry for Discovery Proteomics: Quantifying Translational Cell Heterogeneity in the 16-Cell Frog (Xenopus) Embryo

Significance Statement

Single-cell mass spectrometry provides unique investigative opportunities to decipher cell-to-cell differences by extending discovery (untargeted) molecular measurements to limited amounts of materials. In this original work, Prof. Peter Nemes (PI) and Mrs. Camille Lombard-Banek of the GW Department of Chemistry enhanced the detection sensitivity of high-resolution mass spectrometry to ask with Prof. Sally A. Moody from the GW Department of Anatomy and Regenerative Biology how embryonic cells that give rise to different types of tissues host different proteomes. The research group developed workflows to dissect single embryonic cells from the 16-cell frog (Xenopus laevis) embryo, lyse and extract the proteins from these cells, and digest the proteins for analysis via a bottom-up proteomic workflow. The resulting peptides were bar-coded for cell identity using designer mass tags, pooled, and analyzed using a custom-built single-cell capillary electrophoresis electrospray ionization mass spectrometer. Quantification of ~150 protein groups revealed biologically and statistically significant differences in protein composition between embryonic cells that occupy the animal-vegetal and dorso-ventral axes of the embryo, forming nervous, skin, and gut tissues later during development of the vertebrate embryo. Besides demonstrating sufficient analytical sensitivity to use mass spectrometry for discovery measurements on single cells, the study provided new data for cell and developmental biology on the patterning of the early embryo. In combination with already available data on gene transcription, singe-cell mass spectrometry raises a powerful new opportunity to help better understand the molecular players underlying normal embryonic development. This research was funded by the National Science Foundation Division of Biological Infrastructure (grant no. 1455474).

Figure Legend: Quantification of proteomic differences between single embryonic cells in the 16-cell Xenopus embryo using proteomic single-cell high-resolution mass spectrometry.

About The Author

Peter Nemes holds a PhD in Chemistry from the George Washington University (Washington, DC), where he developed laser ablation electrospray ionization mass spectrometry for in situ and in vivo analysis as well as molecular imaging in two and three dimensions as a PhD graduate student (advisor: Prof. Akos Vertes). He completed postdoctoral training in analytical neuroscience at the University of Illinois—Urbana-Champaign (mentor: Prof. Jonathan V. Sweedler), where he developed mass spectrometry technologies to measure small and large molecules in single neurons and to image their spatial distribution. One of these technologies was single-cell capillary electrophoresis, which revealed metabolomic heterogeneity between different neuron types in the central nervous system of Aplysia californica and adoptability of the single-neuronal metabolome to external conditions. Another technology was a custom-built MALDI-C60-SIMS dual ion source mass spectrometer that helped probe the spatial distribution of small-to-large molecules in single neurons. In 2011, Dr. Nemes joined the Food and Drug Administration (FDA, Silver Spring, MD) as a Principal Investigator, where he developed mass spectrometry-based technologies to enable the high-throughput screening of chemical contaminants in regulated drug products and medical devices. There, he developed a mass spectrometry facility and served as the Laboratory Leader of the Laboratory of Chemical Contamination at the Division of Chemistry and Materials Science. In 2013, Dr. Nemes became an Assistant Professor at the Department of Chemistry of the George Washington University. His research develops high-sensitivity mass spectrometry platforms to assess the spatiotemporal evolution of metabolic and proteomic processes. Current work in the Nemes laboratory elucidates molecular mechanisms by which (i) cells acquire different fates in the developing vertebrate embryo and the central nervous system and (ii) respond to external stimuli such as drugs of treatment and toxins. Prof. Nemes has authored 30 peer-reviewed publications, 6 book chapters, and 80+ presentations, and holds 4 licensed patents.  He received the 2008 International Research Fellowship award by the Dimitris N. Chorafas Foundation (Luzern, Switzerland), the 2009 American Institute of Chemists prize in Chemistry by the American Institute of Chemists (Washington, DC), the 2010 Science and Technology Innovation Award by Baxter Healthcare Corporation (Chicago, IL), the 2011 Special recognition by the FDA (Silver Spring, MD), and the 2016 Arthur Findeis Award for Achievements by a Young Analytical Scientist by the American Chemistry Society. Prof. Nemes is a Beckman Young Investigator by the Arnold and Mabel Beckman Foundation. 

About The Author

Camille Lombard-Banek obtained a Master of Science in chemical engineering from the Ecole Nationale de Chime de Lille (Paris, France) and in chemistry from the University of Toledo (Toledo, OH). She is currently completing her PhD at the George Washington University (Washington, DC) under the tutelage of Prof. Peter Nemes. Her PhD research focuses on advancing mass spectrometry technology to enable the characterization of basic molecular processes in single embryonic cells as they commit to different tissue fates. Mrs. Lombard is recipient of the 2015 Georges Guiochon Student Travel Award from the Washington Chromatography Discussion Group (Washington, DC). 

About The Author

Sally A. Moody received her Ph. D. in Neuroscience from the University of Florida College of Medicine where she studied motor axon guidance cues in the trigeminal system of the chick embryo. She did her postdoctoral work in the Department of Neurobiology and Anatomy at the University of Utah School of Medicine where she studied axon guidance and cell lineage in Xenopus embryos. She was an Assistant and then tenured Associate Professor in the Department of Anatomy and Cell Biology at the University of Virginia School of Medicine, with a joint appointment in the Department of Neuroscience. There she made extensive fate maps of the cleavage stage Xenopus embryos, demonstrated the roles of various extracellular matrix proteins in axon growth, identified a maternal RNA contribution to neural fate and demonstrated lineage influences on the determination of amacrine cell fate. She moved to the George Washington University School of Medicine and Health Sciences in 1992, where she is Professor of Anatomy and Regenerative Medicine. Currently, her laboratory is studying the gene regulatory network that stabilizes neural stem cell fate and the factors that are required for cranial sensory placode development. She has served on the editorial boards of several journals in the fields of neuroscience, developmental and stem cell biology.

 

Journal Reference

Angew Chem Int Ed Engl. 2016 Feb;55(7):2454-8.

Lombard-Banek C¹, Moody SA², Nemes P³.

Show Affiliations

1. Department of Chemistry, W. M. Keck Institute for Proteomics Technology and Applications, The George Washington University, 800 22nd Street, NW, Suite 4000, Washington, DC, 20052, USA.
2. Department of Anatomy and Regenerative Biology, The George Washington University, Washington, DC, 20052, USA.
3. Department of Chemistry, W. M. Keck Institute for Proteomics Technology and Applications, The George Washington University, 800 22nd Street, NW, Suite 4000, Washington, DC, 20052, USA. petern@gwu.edu.

Abstract

We advance mass spectrometry from a cell population-averaging tool to one capable of quantifying the expression of diverse proteins in single embryonic cells. Our instrument combines capillary electrophoresis (CE), electrospray ionization, and a tribrid ultrahigh-resolution mass spectrometer (HRMS) to enable untargeted (discovery) proteomics with ca. 25 amol lower limit of detection. CE-μESI-HRMS enabled the identification of 500-800 nonredundant protein groups by measuring 20 ng, or <0.2% of the total protein content in single blastomeres that were isolated from the 16-cell frog(Xenopus laevis) embryo, amounting to a total of 1709 protein groups identified between n=3 biological replicates. By quantifying ≈150 nonredundant protein groups between all blastomeres and replicate measurements, we found significant translational cell heterogeneity along multiple axes of the embryo at this very early stage of development when the transcriptional program of the embryo has yet to begin.

© 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

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Definition of an 18-mer Synthetic Peptide Derived from the GB virus C E1 Protein as a New HIV-1 Entry Inhibitor

Significance Statement

In this article we report the construction of several peptide libraries scanning the GB virus C (GBV-C) E1 protein and the evaluation of their anti-HIV-1 activity. On the basis of the results, an 18-mer synthetic peptide, specifically the sequence E1(139-156) (namely E1P47), is defined as a new anti-HIV-1 peptide entry inhibitor. The peptide showed similar antiviral activity against viruses from clades A, B, C, D and AE. The broad spectrum of activity against HIV-1 that E1P47 has been demonstrated to have, as well as its capacity to prevent HIV infection by inhibiting (at least partially) viral entry, constitute favourable properties when evaluating this small peptide as a microbicide lead. We consider that these results are of interest for people working in the field of discovering new peptides with the ability of inhibiting HIV and more specifically those involved in studying the relationship of GB virus C and HIV viruses. The results described in the present work indicate the implication of the E1 envelope of the GB virus C in the inhibition of HIV-1. Moreover, our results also highlight the definition of one inhibiting domain within this protein. Thus, taken together the results allow us to consider also the non-pathogenic E1 GB virus C protein as an attractive source of peptides for the development of novel anti-HIV therapies. We strongly believe that the results reported here are important in themselves, so far as we have defined a novel lead compound as in that for the first time the inhibitory role of a highly conserved fragment of the E1 protein is reported.

Group information

The research interests of the Unit of Synthesis and Biomedical Applications of Peptides deal with the use of peptides for the development of new biosensors for the diagnosis of human illnesses and on the selection of therapeutic agents of peptide origin through biophysical testing.

Specifically, in the field of synthetic peptides for the diagnosis and prognosis of rheumatoid arthritis, we aim to identify new antigen peptides, derived from citrullinated/homocitrullinated proteins present in rheumatoid synovial fluid (fibrin, filaggrin, vimentin and enolase) in order to thereby identify those patients who require more aggressive therapies right from the moment of diagnosis of the disease. That would allow greater control of the disease and, consequently, less harm to the articulations and a better prognosis. Moreover, the detection of a single biomarker is not always sufficiently sensitive or precise to discriminate between RA patients with different prognoses, clinical characteristics or responses to therapy. Through the simultaneous analysis of the target peptides, incorporated into a multiplex test, we aim to facilitate the biological fingerprinting of autoantibodies in serum that will be able to identify subgroups of patients with specific clinical characteristics; different prognoses; and who either respond well to, or suffer negative effects from, certain therapeutic interventions. These results could have far-reaching practical implications for establishing the most appropriate therapeutic strategy for RA patients.

Related with the study of peptide human immunodeficiency virus entry inhibitors, we propose the study of new therapeutic agents for the treatment of AIDS, together with the development of a biosensor platform that can be used to detect antibodies against GB virus C in patients co-infected with HIV-1.

Finally, the objective of the research line on peptide controlled-release nanosystems for ocular administration of drugs includes the development of new systems of administration based on liposomes and nanoparticles that are targeted via the use of peptides and that can ensure low levels of irritation, sufficient bioavailability and compatibility with ocular tissues.

About The Author

Prof. Isabel Haro. Senior Staff. B.Sc. Chemistry, University of Barcelona, 1982. M.Sc. Biochemistry,University of Barcelona, 1984. Ph.D. Chemistry, University of Barcelona, 1988. Postdoctoral fellow (1989-1990) between CSIC in Barcelona and Royal Free Hospital in London (Prof. G. Gregoriadis). Research Scientist at CSIC leading the Unit of Synthesis and Biomedical Applications of CSIC in 1990. Since then she has been working in the use of synthetic peptides in the field of biomedicine, specifically in the development of new biosensors for the diagnosis of human illnesses and in the selection of new therapeutic agents of peptide origin through biophysical testing. She has published more than 180 articles, 8 reviews, 30 chapters of books and 8 patents. Also, she has been the principal researcher of 15 research projects and 3 contracts with the industry and supervised 12 doctoral theses. e-mail contact: isabel.haro@iqac.csic.es  

About The Author

Dr. Maria JoséGomara. Senior Staff. B.Sc. Pharmacy, University of Barcelona, 1992. M.Sc. Physical Chemistry, University of Barcelona, 1995. Ph.D. Pharmacy, University of Barcelona, 2000. Postdoctoral fellow (2001-2004) in Prof. Felix Goñi laboratory (Biophysical Unit, CSICEHU/UPV). Research assistant (2005-2007) in the Unit of Synthesis and Biomedical Applications of Peptides leaded by Dr. Haro, IQAC-CSIC. Research scientist of CSIC since 2008. Experience in peptide synthesis, biophysical studies of peptide-lipid and peptide-peptide interactions and development of peptide-based immunoassays. She has published over 60 articles and 5 patents. e-mail contact: mariajose.gomara@iqac.csic.es 

Journal Reference

Biochim Biophys Acta. 2016 Feb 22;1860(6):1139-1148.

Gómara MJ¹, Sánchez-Merino V², Paús A³, Merino-Mansilla A², Gatell JM⁴, Yuste E², Haro I⁵.

Show Affiliations

1. Unit of Synthesis and Biomedical Application of Peptides. IQAC-CSIC, Jordi Girona, 18-26, 08034 Barcelona, Spain. Electronic address: mariajose.gomara@iqac.csic.es.
2. AIDS Research Unit, Institut d’Investigacions Biomèdiques August Pi I Sunyer, Barcelona, Spain. HIVACAT, Barcelona, Spain.
3. Unit of Synthesis and Biomedical Application of Peptides. IQAC-CSIC, Jordi Girona, 18-26, 08034 Barcelona, Spain.
4. AIDS Research Unit, Institut d’Investigacions Biomèdiques August Pi I Sunyer, Barcelona, Spain. HIVACAT, Barcelona, Spain; Infectious Diseases Unit-HIVACAT, Hospital Clinic, Villarroel, 170, 08036 Barcelona, Spain.
5. Unit of Synthesis and Biomedical Application of Peptides. IQAC-CSIC, Jordi Girona, 18-26, 08034 Barcelona, Spain. Electronic address: isabel.haro@iqac.csic.es.

Abstract

BACKGROUND:

A slower progression of AIDS and increased survival in GB virus C positive individuals, compared with GB virus C negative individuals has been demonstrated; while the loss of GB virus C viremia was closely associated with a rise in mortality and increased progression of AIDS. Following on from the previous reported studies that support the thesis that GB virus C E2 interferes with HIV-1 entry, in this work we try to determine the role of the GB virus C E1 protein in HIV-1 inhibition.

METHODS:

The present work involves the construction of several overlapping peptide libraries scanning the GBV-C E1 protein and the evaluation of their anti-HIV activity.

RESULTS:

Specifically, an 18-mer synthetic peptide from the GB virus C E1 protein, E1(139-156), showed similar antiviral activity against HIVs from viruses from clades A, B, C, D and AE. Competitive ELISA using specific gp41-targeting mAbs, fluorescence resonance energy transfer as well as haemolysis assays demonstrated that this E1 peptide sequence interacts with the highly conserved N-terminal region of the HIV-1 gp41 (the fusionpeptide) which is essential for viral entry.

CONCLUSIONS:

We have defined a novel peptide lead compound and described the inhibitory role of a highly conserved fragment of the E1 protein.

GENERAL SIGNIFICANCE:

The results together allow us to consider the non-pathogenic E1 GB virus C protein as an attractive source of peptides for the development of novel anti-HIV therapies.

Copyright © 2016 Elsevier B.V. All rights reserved.

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Trace analysis of three fungicides in animal origin foods with a modified QuEChERS method and liquid chromatography-tandem mass spectrometry

Significance Statement

Most people ignore the risk of pesticide residues in biological materials of animal origin, which makes people more vulnerable to some potential epidemics. However, plant materials, such as straws, grains and leaves, which used for feeding livestock may be contaminated, and, consequently, pesticides may be consumed by humans via animal feed. Due to the lipophilic nature of some pesticides, they are accumulated in milk, meat or animal offal and enter the human body through the food chain, and then may cause serious health problems. Increasing attention has been paid to the risks posed to consumers by pesticide residues in animal foods. However, as far as we know, few articles referred to the pesticide detection in animal origin foods. Amongst commercial pesticides, propiconazole , pyraclostrobin and isopyrazam , three of most widely used fungicides in the world, deserves special attention because of its high effectiveness against diseases affecting cereals, fruits, and vegetables. The key of our study is to use the same method to achieve simultaneous detection for the three fungicides in seven animal origin foods, which will save labor, time and cost significantly and make it more suitable for the detection of large-scale real samples.

Journal Reference

Anal Bioanal Chem. 2016 Feb;408(5):1515-22.

Mu Z¹, Feng X¹, Zhang Y¹, Zhang H².

Show Affiliations

1. College of Science, China Agricultural University, Beijing, 100193, China.
2. College of Science, China Agricultural University, Beijing, 100193, China. hongyan@cau.edu.cn.

Abstract

A multi-residue method based on modified QuEChERS (quick, easy, cheap, effective, rugged, and safe) sample preparation, followed by liquid chromatography tandem mass spectrometry (LC-MS/MS), was developed and validated for the determination of three selected fungicides (propiconazole, pyraclostrobin, and isopyrazam) in seven animal origin foods. The overall recoveries at the three spiking levels of 0.005, 0.05, and 0.5 mg kg(-1) spanned between 72.3 and 101.4 % with relative standard deviation (RSD) values between 0.7 and 14.9 %. The method shows good linearity in the concentrations between 0.001 and 1 mg L(-1) with the coefficient of determination (R (2)) value >0.99 for each target analyte. The limit of detections (LODs) for target analytes were between 0.04 and 1.26 μg kg(-1), and the limit of quantifications (LOQs) were between 0.13 and 4.20 μg kg(-1). The matrix effect for each individual compound was evaluated through the study of ratios of the areas obtained in solvent and matrix standards. The optimized method provided a negligible matrix effect for propiconazole within 20 %, whereas for pyraclostrobin and isopyrazam, the matrix effect was relatively significant with a maximum value of 49.8 %. The developed method has been successfully applied to the analysis of 210 animal origin samples obtained from 16 provinces of China. The results suggested that the developed method was satisfactory for trace analysis of three fungicides  in animal  origin foods. Graphical abstract Schematic representation of the proposed residue analytical  method for  animal  origin foods using  QuEChERS and LC-MS/MS.

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Dual effects of acetylsalicylic acid on ERK signaling and Mitf transcription lead to inhibition of melanogenesis

Significance Statement

Non-steroidal anti-inflammatory drugs (NSAIDs) are known to act by directly suppressing the activity of cyclooxygenase, the key enzyme catalyzing the biosynthesis of prostaglandin, which induce inflammation.  Therefore, NSAIDs are usually used for treat pain, inflammation, and fever.  Acetylsalicylic acid (Aspirin) is the most prominent NSAIDs and it is available over-the-counter all over the world.  Also, aspirin has been shown to be effective as an antiplatelet drug in preventing heart attacks, stroke, and cerebral thrombosis.

Skin pigmentation results from melanin synthesis by melanocytes and is caused greater by exposure to UV radiation.  In melanin synthesis, there is a crucial enzyme called tyrosinase that catalyzes chemical reaction: amino acid tyrosine to melanin.  Therefore, mutation of the tyrosinase gene causes albinism.

In this study, we investigate the anti-melanogenic effect of aspirin on aspects of the regulation of melanogenic genes and intracellular pathways.  We focused on microphthalmia-associated transcription factor (Mitf) that is an important factor of melanin synthesis.  Once activated, Mitf promotes gene transcription by binding the promoter region of tyrosinase gene.  Interestingly, aspirin inhibited Mitf gene at the transcriptional level.  The fact that down-regulation of Mitf imply that aspirin depresses the tyrosinase gene and subsequent inhibition of melanogenesis.  Furthermore, we investigate the effect of aspirin on the extracellular signal regulated kinase (ERK) pathway.  ERK plays an important role in cellular processes such as survival, proliferation, cell cycle progression and melanin synthesis.  Activated ERK can phosphorylate Mitf, which causes degradation of Mitf, and this process also brings about inhibition of melanin synthesis.  Surprisingly, our result demonstrated that aspirin activated the ERK and it is also a leading cause of inhibition of melanogenesis.  Taken together, our results suggest that aspirin can inhibit melanin synthesis that is caused by both inhibition of Mitf gene expression and ERK activation.

About The Author

Kazuomi Sato, Ph.D.

Laboratory of Biochemistry, Department of Life Science, Tamagawa University

2009: Received his Ph.D. in Agriculture at Gifu University (Gifu, Japan)

2009-2013: Assistant Professor, Tamagawa University (Tokyo, Japan)

2014-present:  Associate Professor, Tamagawa University (Tokyo, Japan)

Research interests: Regulation of melanin synthesis, Enzyme kinetics, Mechanism of cell death.  

Journal Reference

Mol Cell Biochem. 2016 Jan;412(1-2):101-10. 

Nishio T¹, Usami M², Awaji M², Shinohara S², Sato K^3,4.

Show Affiliations

1. Graduate School of Agriculture, Tamagawa University, 6-1-1 Tamagawa-gakuen, Machida, Tokyo, 194-8610, Japan.
2. Department of Life Science, College of Agriculture, Tamagawa University, 6-1-1 Tamagawa-gakuen, Machida, Tokyo, 194-8610, Japan.
3. Graduate School of Agriculture, Tamagawa University, 6-1-1 Tamagawa-gakuen, Machida, Tokyo, 194-8610, Japan. kzsato@agr.tamagawa.ac.jp.
4. Department of Life Science, College of Agriculture, Tamagawa University, 6-1-1 Tamagawa-gakuen, Machida, Tokyo, 194-8610, Japan. kzsato@agr.tamagawa.ac.jp.

Abstract

Acetylsalicylic acid (ASA) is widely used as an analgesic/antipyretic drug. It exhibits a wide range of biological effects, including preventative effectsagainst heart attack and stroke, and the induction of apoptosis in various cancer cells. We previously found that ASA inhibits melanogenesis in B16 melanoma cells. However, the mechanisms of how ASA down-regulates melanin synthesis remain unclear. Here, we investigated the effect of ASA on melanogenic pathways, such as extracellular signal-regulated kinase (ERK) and microphthalmia-associated transcription factor (Mitf) transcription. ASA significantly inhibited melanin synthesis in a dose-dependent manner without oxidative stress and cell death. Semi-quantitative reverse transcription-polymerase chain reaction analysis showed that the inhibitory effect of ASA might be due to the inhibition of Mitf gene transcription. Interestingly, ASA also induced ERK phosphorylation. Additionally, treatment with PD98059, a specific ERK phosphorylation inhibitor, abolished the anti-melanogenic effect of ASA. These results suggest that the depigmenting effect of ASA results from down-regulation of Mitf, which is induced by both the induction of ERK phosphorylation and the inhibition of Mitf transcription.

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Cerebral mast cells contribute to postoperative cognitive dysfunction by promoting blood brain barrier disruption

About The Author

Yanning Qian, Director of Department of Anesthesiology Research, First affiliated hospital of Nanjing Medical University, Deputy Director of the General Surgery Department of Nanjing Medical University. Research interests: anesthetic effects on nerve-endocrine-immune network. This research direction have had some effect in the domestic and foreign counterparts: achieved the provincial science and technology progress third class Prize 2 times; achieved 3 National Natural Science Foundation; in 2011, achieved one of the Ministry of education doctoral project; published more than 20 papers in SCI journals as first or corresponding author; several papers presented in international conferences. 

Journal Reference

Behav Brain Res. 2016 Feb 1;298(Pt B):158-66.

Zhang S¹, Dong H¹, Zhang X¹, Li N¹, Sun J², Qian Y³.

Show Affiliations

1. Department of Anesthesiology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, PR China.
2. Department of Anesthesiology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, PR China. Electronic address: dgsunjie@hotmail.com.
3. Department of Anesthesiology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, PR China. Electronic address: yanning_qian@163.com.

Abstract

Trauma induced neuroinflammation plays a key role in the development of  postoperative  cognitive dysfunction  . The blood-brain barrier (BBB), a highly specialized endothelial layer, is exquisitely sensitive to inflammatory insults, which can result in numerous neurocognitive syndromes. While brain mast cells are the “first responder” in the injury, the functional interactions between mast cells and the BBB remain poorly understood. Our results demonstrate that tibial fracture surgery can induce cognitive impairment relating to an inflammatory response and destabilization of the BBB. Disodium cromoglycate (cromolyn) – which acts as a mast cell stabilizer – inhibited this effect. Specifically, cromolyn resulted in ameliorated cognitive ability, decrease of inflammatory cytokines and increase of BBB stability. Taken together, these results suggest that activated mast cells contributed to central nervous system inflammation and cognitive dysfunction by promoting BBB disruption, and interactions between mast cells and the BBB could constitute a new and unique therapeutic target for postoperative cognitive dysfunction.

Copyright © 2015 Elsevier B.V. All rights reserved.

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Central amygdala lesions inhibit pontine nuclei acoustic reactivity and retard delay eyeblink conditioning acquisition in adult rats

 Journal Reference

Learn Behav. 2015 Oct 20. 

Pochiro JM¹, Lindquist DH^2,3.

Show Affiliations

1. Department of Neuroscience, The Ohio State University, 1835 Neil Avenue, Room 49, Columbus, OH, 43210, USA.
2. Department of Psychology, The Ohio State University, 1835 Neil Avenue, Room 49, Columbus, OH, 43210, USA. lindquist.40@osu.edu.
3. Department of Neuroscience, The Ohio State University, 1835 Neil Avenue, Room 49, Columbus, OH, 43210, USA. lindquist.40@osu.edu.

Abstract

In delay eyeblink conditioning (EBC) a neutral conditioned stimulus (CS; tone) is repeatedly paired with a mildly aversive unconditioned stimulus (US; periorbital electrical shock). Over training, subjects learn to produce an anticipatory eyeblink conditioned response (CR) during the CS, prior to US onset. While cerebellar synaptic plasticity is necessary for successful EBC, the amygdala is proposed to enhance eyeblink CR acquisition. In the current study, adult Long-Evans rats received bilateral sham or neurotoxic lesions of the central nucleus of the amygdala (CEA) followed by 1 or 4 EBC sessions. Fear-evoked freezing behavior, CS-mediated enhancement of the unconditioned response (UR), and eyeblink CR acquisition were all impaired in the CEA lesion rats relative to sham controls. There were also significantly fewer c-Fos immunoreactive cells in the pontine nuclei (PN)-major relays of acoustic information to the cerebellum-following the first and fourth EBC session in lesion rats. In sham rats, freezing behavior decreased from session 1 to 4, commensurate with nucleus-specific reductions in amygdala Fos+ cell counts. Results suggest delay EBC proceeds through three stages: in stage one the amygdala rapidly excites diffuse fear responses and PN acoustic reactivity, facilitating cerebellar synaptic plasticity and the development of eyeblink CRs in stage two, leading, in stage three, to a diminution or stabilization of conditioned fear responding.

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Role of iso-receptors in receptor-receptor interactions with a focus on dopamine iso-receptor complexes

Significance Statement

The discovery of receptor-receptor interactions (RRIs) in the early 1980s together with a more accurate focusing of allosteric mechanisms in proteins, significantly expanded the knowledge on the G protein-coupled receptors (GPCR)-mediated signaling processes in the cells. In particular, increasing evidence was provided that GPCR operate not only as monomers but also as quaternary structures (homodimers, heterodimers and higher-order oligomers), in which networks of electrostatic interactions (hydrogen bonds, van der Waals forces) shape the configuration of the single receptors and the topology of the entire complex, allowing an integration of the incoming signals already at the plasma membrane level through allosteric receptor-receptor interactions. Once established, these integrative mechanisms change the function of the involved GPCR, leading to a sophisticated dynamics of the receptor assembly in terms of modulation of recognition and signaling.

As a consequence, when the interacting receptors are iso-receptors a single neurotransmitter becomes able to induce a spectrum of possible cellular responses that can either work in parallel (redundancy) or interact with each other (complex responses). These factors lead to an astonishing increase in the modes for the recognition/decoding processes that control the cellular biochemical machineries and appear of particular interest for possible therapeutic applications, as indicated by studies on the dopamine iso-receptor complexes D1-D2 and D1-D3 suggesting them as possible targets for neuropsychiatric disorders.

Thus, the expansion of the GPCR field emerging from the characterization of receptor-receptor interactions can also pave the way for the development of novel pharmacological strategies for the treatment of several pathologies, and in the next future this research effort is likely to have a major impact on molecular medicine.

Figure Legend: Micro-domains of the plasma membrane where Receptor-Receptor Interactions (RRIs) can lead to the formation of receptor complexes (Receptor Mosaics, RMs).  From the topology and dynamics of these receptor assemblies a wide spectrum of possible cellular responses to an incoming signal can emerge (Agnati, Guidolin et al., Prog Neurobiol, 2010)

Journal Reference

Rev Neurosci. 2016 Jan 1;27(1):1-25.

Luigi F. Agnati¹ , Diego Guidolin² , Chiara Cervetto³ , Dasiel O. Borroto-Escuela⁴ , Kjell Fuxe⁴ 

Show Affiliations

1. Department of Biomedical Sciences, University of Modena and Reggio Emilia, Modena, Via Campi 287, 41100 Modena, Italy
2. Department of Molecular Medicine, University of Padova, Via Gabelli 65, 35121 Padova, Italy
3. Department of Pharmacy, University of Genova, Viale Cembrano 4, 16147 Genova, Italy
4. Department of Neuroscience, Karolinska Institutet, Retzius vag 8, 17177 Stockholm, Sweden

Abstract

Intercellular and intracellular communication processes consist of signals and recognition/decoding apparatuses of these signals. In humans, the G protein-coupled receptor (GPCR) family represents the largest family of cell surface receptors. More than 30 years ago, it has been proposed that GPCR could form dimers or higher-order oligomers (receptor mosaics [RMs] at the plasma membrane level and receptor-receptor interactions [RRIs] have been proposed as a new integrative mechanism for chemical signals impinging on cell plasma membranes). The basic phenomena involved in receptor-receptor interactions  are allostery and cooperativity of membrane receptors, and the present paper provides basic information concerning their relevance for the integrative functions of RMs. In this context, the possible role of iso-receptor RM is discussed (with a special focus on dopamine receptor subtypes and on some of the RMs they form with other dopamine iso-receptors), and it is proposed that two types of cooperativity, namely, homotropic and heterotropic cooperativity, could allow distinguishing two types of functionally different RMs. From a general point of view, the presence of iso-receptors and their topological organization within RMs allow the use of a reduced number of signals for the intercellular communication processes, since the target cells can recognize and decode the same signal in different ways. This theoretical aspect is further analyzed here by means of an analogy with artificial information systems. Thus, it is suggested that the ‘multiplexer’ and ‘demultiplexer’ concepts could, at least in part, model the role of RMs formed by iso-receptors in the information handling by the cell.

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Precision Pulse Capsulotomy: Preclinical Safety and Performance of a New Capsulotomy Technology

Significance Statement

The ability to reproducibly automate a perfectly sized and circular capsulotomy has spurred much of the current clinical interest in femtosecond laser assisted cataract surgery (FLACS). Compared to manual continuous curvilinear capsulorhexis (CCC), the femtosecond laser capsulotomy (FSLC) is highly reproducible, uniformly more circular, and has a more precise diameter. However, this comes at a much higher capital and procedural cost and disrupts the normal surgical workflow because the FLACS steps cannot be performed within the usual operative sequence. Furthermore, differing national regulations may ban or restrict the ability to balance bill patients for the additional costs associated with FLACS. An important concern has arisen from published reports of an increased rate of anterior capsule tears following FSLC^1,2. Scanning electron microscopy (SEM) of FSLC anterior capsule buttons demonstrates a rougher edge, when compared to manual CCC specimens. In addition, SEM analysis also reveals scattered aberrant laser shots that may be explained by microscopic eye movements occurring during the FSLC step¹. These might be postulated to predispose focal areas of the anterior capsular rim to radial tears caused by subsequent surgical forces.

Mynosys (Fremont, California) has developed a novel capsulotomy method and technology called Precision Pulse Capsulotomy (PPC) and trade named Zepto. A disposable handpiece and nano-engineered capsulotomy tip are powered by a small console to automatically and instantaneously create a perfectly circular capsulotomy of a precise pre-designed diameter. The tip consists of a circular nitinol ring surrounded by a thin silicone cover shaped like a miniature inverted frying pan. Nitinol is a shape memory alloy which means that a 5.0 mm diameter ring can be compressed and deformed for insertion through a clear corneal incision, but will then re-assume its natural circular shape once it emerges inside the anterior chamber (AC). After filling the AC with ophthalmic viscosurgical device (OVD), a retractable metal push rod elongates the ring and silicone shell into a narrower profile that can be inserted through the clear corneal incision. After retracting this push rod, the compressed tip resumes its native circular shape within the AC.

The surgeon gently positions the ring and surrounding suction cup onto the anterior capsular surface prior to applying a small amount of suction via the external console. Only slight suction is needed to appose the anterior capsule against the bottom edge of the nitinol ring, which has been precisely engineered at the micron scale to enable uniform capsule cutting. A rapid series of microsecond-long electrical pulses is used to create the capsulotomy. Phase transition of water molecules trapped between the capsule and nitinol edge cause the stretched capsular membrane to abruptly split circumferentially all at once. Unlike the sequential circular path of a manual or FSLC, the PPC technology mechanically and simultaneously cleaves all 360 degrees of the apposed capsule without cauterizing it. Collateral ocular tissue safety is achieved through two design features. First, the application of energy is extremely brief and confined only to the microscopic edge of the nitinol ring. Second, during activation the nitinol ring is completely covered by the silicone suction cup and further insulated by the surrounding OVD.

This device has been developed through extensive testing in animal and human cadaver eyes³. Miyake Apple view video imaging shows insignificant zonular traction while performing PPC in both rabbit and human cadaver eyes. Pre-clinical performance and safety testing in live rabbits, including intraoperative thermocouple measurements, slit lamp evaluation and histopathology, has been performed at the Moran Eye Center.

On SEM of human cadaver capsules, the PPC capsulotomy edge is smoother and more defect-free than that of a manual CCC.  We have performed extensive testing of capsulotomy edge strength by comparing PPC to both manual CCC and FSLC in paired fellow human cadaver eyes. The PPC edge is consistently stronger than either alternative.

The obvious potential advantage of Zepto PPC would be its ability to reproduce automate the capsulotomy step with a disposable instrument that is inserted in the conventional surgical sequence and in lieu of using capsulorhexis forceps. Surgeons would welcome a method to assure a perfectly sized and round capsulotomy without the workflow challenges and increased procedural time of FLACS. As with FSLC, popular indications would be for complicated cases or when using premium refractive IOLs. The lower cost should ideally make PPC available to all patients independent of affordability and regional regulations regarding balance billing. Surgeons might also opt for the efficiency of using Zepto routinely rather than only in select cases, particularly if it is on average faster and more consistent than manual capsulorhexis.

Because it is an integrated step during conventional phacoemulsification, PPC can be performed after insertion of iris expansion devices for small pupils. The tip is also designed to allow insertion through a smaller pupil if necessary. It does not require additional surgical steps such as capsular staining. If the PPC edge is indeed more tear resistant, this might improve surgical safety by reducing anterior and posterior capsular tears. Finally, the transparent silicone suction cup has a central window that is designed to permit patient fixation on the microscope light filament during positioning of the device. Being able to center the capsulotomy on the visual axis would be advantageous when implanting refractive lens implants such as toric and multifocal IOLs.

The PPC device name was chosen because in the metric scale, zepto is one million times smaller than femto. Both the small size of the instrument and the several millisecond speed of capsulotomy creation inspired this name. The device is CE mark approved, and has been submitted for FDA 510(k) approval.

REFERENCES:

1. Abell RG, Davies PE, Phelan D, et al. Anterior capsulotomy integrity after femtosecond laser-assisted cataract surgery. Ophthalmology 2014;121:17-24.
2. Chang JS, Chen IN, Chan WM, et al. Initial evaluation of a femtosecond laser system in cataract surgery. J Cataract Refract Surg 2014;40:29-36.
3. Chang DF, Mamalis N, Werner L. Precision Pulse Capsulotomy – Preclinical Safety and Performance of a New Capsulotomy Technology. Ophthalmology 2016; 123:255-264.
4. Thompson VM, Berdahl JP, Solano JM, Chang DF. Comparison of manual, femtosecond laser, and precision pulse capsulotomy edge tear in paired human cadaver eyes. Ophthalmology 2016;123:265-274.

Dr. Chang is clinical professor at the University of California, San Francisco. His relevant financial disclosure is that he has received compensation as a consultant for Mynosys, AMO, and LensAR. 

About The Author

David F. Chang, MD is a Summa Cum Laude graduate of Harvard College and Harvard Medical School. He completed his ophthalmology residency at the University of California, San Francisco where he is now a clinical professor. Dr. Chang has been on the ASCRS Board and Executive Committee since 2009 and served as the 2012-2013 president. He is immediate past chair of the AAO Cataract PPP Panel and in 2009 completed his 5-year term as chair of the AAO Annual Meeting Program Committee. Dr. Chang also co-chairs the ASCRS Foundation. He has served as chief medical editor of EyeWorld and co-chief medical editor for Cataract and Refractive Surgery Today, In 2006, Dr. Chang became only the third ophthalmologist to ever receive the Charlotte Baer Award honoring the outstanding clinical faculty member at the UCSF Medical School. He has received the highest honor for cataract surgery or for an international ophthalmologist from the following organizations: ASCRS (Binkhorst Medal), AAO (Kelman Lecture), Asia Pacific Association of Cataract & Refractive Surgery (Lim Medal), United Kingdom and Ireland Society of Cataract & Refractive Surgery (Rayner Medal), Canadian Society of Cataract and Refractive Surgery (Award of Excellence/Stein Lecture), All India Ophthalmology Society (President’s Lecture), Indian Intraocular Implant & Refractive Society (Gold Medal), Italian Ophthalmological Society (Strampelli Medal), Royal Australia and New Zealand College of Ophthalmologists (Gregg Medal), and the Asia-Pacific Academy of Ophthalmology (Jose Rizal International Medal). In 2014, he was also voted the 5^th most influential ophthalmologist in the world by the international readership of The Ophthalmologist.

Journal Reference

Ophthalmology. 2016 Feb;123(2):255-64.

Chang DF¹, Mamalis N², Werner L².

Show Affiliations

1. Altos Eye Physicians, Los Altos, California. Electronic address: dceye@earthlink.net.
2. John A. Moran Eye Center, University of Utah, Salt Lake City, Utah.

Abstract

PURPOSE:

To assess the preclinical safety and performance of a new precision pulse capsulotomy (PPC) method.

DESIGN:

Human cadaver eye studies and surgical, slit-lamp, and histopathologic evaluation in a consecutive series of 20 live rabbits.

PARTICIPANTS:

Human cadaver eyes and New Zealand white rabbits.

METHODS:

Precision pulse capsulotomy uses a highly focused, fast, multipulse, low-energy discharge to produce a perfectly round anteriorcapsulotomy instantaneously and simultaneously along all 360°. Capsulotomies are performed using a disposable handpiece with a soft collapsible tip and circular nitinol cutting element. Miyake-Apple imaging and scanning electron microscopy (SEM) of PPC were conducted in human cadaver eyes. Surgical, postoperative slit-lamp, and histopathologic assessments of PPC were performed in 20 live rabbits and were compared with manual continuous curvilinear capsulorrhexis (CCC) in the fellow eye. Anterior chamber (AC) thermocouple temperature measurements were evaluated in a subset of rabbit eyes.

MAIN OUTCOME MEASURES:

Capsulotomy edge circularity, SEM morphologic features and zonular movement with PPC in human cadaver eyes. Anterior chamber temperature during PPC and grading of ocular inflammation, corneal endothelial damage, anterior capsular opacification (ACO), and posterior capsular opacification (PCO).

RESULTS:

Miyake-Apple imaging showed minimal zonular stress, and thermocouple measurements demonstrated negligible AC temperature changes during PPC. Precision pulse capsulotomy produced round, complete capsulotomies in all 20 rabbit eyes, leading to successful in-the-bag intraocular lens (IOL) implantation. Slit-lamp examinations at 3 days and 1, 2, and 4 weeks after surgery showed no significant differences between PPC and CCC in corneal edema, AC inflammatory reaction, capsular fibrosis, ACO, and PCO. Postmortem studies showed no difference in the corneal endothelium between PPC and CCC eyes. All IOLs were well centered in PPC eyes, and histopathologic analysis showed no greater inflammatory infiltrates.

CONCLUSIONS:

Precision pulse capsulotomy is a new method to automate consistent creation of a perfectly circular anterior capsulotomy with a disposable handheld instrument that can be used in the normal phacoemulsification surgical sequence. Compared with CCC in fellow rabbit eyes, PPC was equally safe and showed no greater zonular stress compared with CCC in human cadaver eyes. Human cadaver eye SEM showed a much smoother capsulotomy edge compared to those produced by femtosecond laser.

Copyright © 2016 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.

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Acoustofluidic particle manipulation inside a sessile droplet: four distinct regimes of particle concentration

Journal Reference

Lab Chip. 2016 Feb 21;16(4):660-7.

Destgeer G, Cho H, Ha BH, Jung JH, Park J, Sung HJ.

Department of Mechanical Engineering, KAIST, Daejeon 34141, Korea. hjsung@kaist.ac.kr.

Abstract

In this study, we have investigated the motion of polystyrene microparticles inside a sessile droplet of water actuated by surface acoustic waves (SAWs), which produce an acoustic streaming flow (ASF) and impart an acoustic radiation force (ARF) on the particles. We have categorized fourdistinct regimes (R1-R4) of particle aggregation that depend on the particle diameter, the SAW frequency, the acoustic wave field (travelling or standing), the acoustic waves’ attenuation length, and the droplet volume. The particles are concentrated at the centre of the droplet in the form of a bead (R1), around the periphery of the droplet in the form of a ring (R2), at the side of the droplet in the form of an isolated island (R3), and close to the centre of the droplet in the form of a smaller ring (R4). The ASF-based drag force, the travelling or standing SAW-based ARF, and the centrifugal force are utilized in various combinations to produce these distinct regimes. For simplicity, we fixed the fluid volume at 5 μL, varied the SAW actuation frequency (10, 20, 80, and 133 MHz), and tested several particle diameters in the range 1-30 μm to explicitly demonstrate the regimes R1-R4. We have further demonstrated the separation of particles (1 and 10 μm, 3 and 5 μm) using mixed regime configurations (R1 and R2, R2 and R4, respectively).

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