Tamper-proof tablets for distinction between counterfeit and originator drugs through PEG coding

Eur J Pharm Biopharm. 2016 Feb;99:1-6.

Ilko D¹, Steiger C¹, Keller R², Holzgrabe U³, Meinel L¹.

Show Affiliations

¹Institute for Pharmacy and Food Chemistry, University of Wuerzburg, Am Hubland, DE-97074 Wuerzburg, Germany.

²Kliniken der Stadt Köln, Zentrallabor, Ostheimerstr. 200, 51109 Köln, Germany.

³Institute for Pharmacy and Food Chemistry, University of Wuerzburg, Am Hubland, DE-97074 Wuerzburg, Germany. Electronic address: ulrike.holzgrabe@uni-wuerzburg.de.

 

Abstract

Counterfeit drugs are a major threat to public health. Current efforts focus on serialization of the secondary packaging which do not allow to trace the individual unit. As a proof of concept, we intended to mark each tablet for its unambiguous recognition. Spiking monodisperse PEGs into tablet coating solutions at concentrations as low as 3 ppm was instrumental to “write” a code into each tablet film which was readily read upon isolation and LC–MS/MS analysis. Different qualities and amounts of monodisperse polyethylene glycols can be used for coding solid drug products. The approach is limited to cases in which PEGs are not present for formulation purposes as excipients, as coding against this background was unfeasible.

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Abrogation of Age-Induced MicroRNA-195 Rejuvenates the Senescent Mesenchymal Stem Cells by Reactivating Telomerase

Journal Reference

Stem Cells. 2016 Jan;34(1):148-59.

Okada M, Kim HW, Matsu-Ura K, Wang YG, Xu M, Ashraf M.

Department of Pathology and Lab of Medicine, University of Cincinnati Medical Center, Cincinnati, Ohio, 45267, USA.

Abstract

Previously, we reported that a novel subpopulation of young mesenchymal stem cells (YMSCs) existed in old bone marrow, which possessed high antiaging properties as well as excellent efficacy for cardiac repair. MicroRNAs (miRNAs) have emerged as key regulators in post-transcriptional gene expression programs, and however, it is unknown whether miRNAs directly control stem cell senescence. Here we present the first evidence that miR-195 overexpressed in old MSCs (OMSCs) induces stem cell senescence deteriorating their regenerative ability by directly deactivating telomerase reverse transcriptase (Tert), and abrogation of miR-195 can reverse stem cell aging. MiRNAs profiling analysis in YMSCs and OMSCs by microarray showed that miR-140, miR-146a/b, and miR-195 were significantly upregulated in OMSCs, which led us to hypothesize that these are age-induced miRNAs involved in stem cell senescence. Of these miRNAs, we found miR-195 directly targeted 3′-untranslated region of Tert gene by computational target prediction analysis and luciferase assay, and knockdown of miR-195 significantly increased Tert expression in OMSCs. Strikingly, miR-195 inhibition significantly induced telomere relengthening in OMSCs along with reduced expression of senescence-associated β-galactosidase. Moreover, silencing miR-195 in OMSCs by transfection of miR-195 inhibitor significantly restored antiaging factors expression including Tert and Sirt1 as well as phosphorylation of Akt and FOXO1. Notably, abrogation of miR-195 markedly restored proliferative abilities in OMSCs. Transplantation of OMSCs with knocked out miR-195 reduced infarction size and improved LV function. In conclusion, rejuvenation of aged stem cells by miR-195 inhibition would be a promising autologous therapeutic strategy for cardiac repair in the elderly patients.

© 2015 AlphaMed Press.

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Thymidine 5′-O-monophosphorothioate induces HeLa cell migration by activation of the P2Y6 receptor

Significance Statement

Ligands of the P2Y nucleotide receptors are of potential interest for pharmacotherapies directed at neurodegenerative disorders, intestinal function, angiogenesis, cardiac remodeling,  etc.  Activation of P2Y6 by its native ligand UDP or by synthetic agonists is associated with vasoconstriction and cytoprotection. P2Y6 seems to control chloride secretion by epithelium, the direct contraction and endothelium-dependent relaxation of the aorta or insulin secretion as well. Pharmacological modulation of the P2Y6 receptor is of increasing interest, nevertheless UDP as a natural ligand was reported to activate not only P2Y6 but also P2Y14. Therefore, agonists that delineate between these two subtypes are needed. Besides, the therapeutic potential of extracellular nucleotides is limited because they are degraded by extracellular enzymes which reduce their efficacy and duration of action. We pay particular attention to the unique activity of thymidine 5’-O-monophosphorothioate (TMPS) which acts as a specific partial agonist of the P2Y6 receptor. Its  increased stability as compared to their unmodified counterpart as well as their affinity towards P2Y6 is especially interesting and may be responsible for some long-term effects.  

About The Author

Edyta Gendaszewska-Darmach:

PhD degree in Bioorganic Chemistry from Polish Academy of Sciences.

an Asissstant Professor at Faculty of Biotechnology and Food Sciences (Institute of Technical Biochemistry),  Lodz University of Technology.

Research involves anti-diabetic and wound healing properties of natural and modified biophosphates (nucleotides and lysophospholipids) as well as biomaterials for regenerative medicine.

Journal Reference

Purinergic Signal. 2016 Jan 8.

Gendaszewska-Darmach E¹, Szustak M².

Show Affiliations

1. Institute of Technical Biochemistry, Faculty of Biotechnology and Food Sciences, Lodz University of Technology, Stefanowskiego 4/10, 90-924, Lodz, Poland. edarmach@wp.pl.
2. Institute of Technical Biochemistry, Faculty of Biotechnology and Food Sciences, Lodz University of Technology, Stefanowskiego 4/10, 90-924, Lodz, Poland.

Abstract

ATP, ADP, UTP, and UDP acting as ligands of specific P2Y receptors activate intracellular signaling cascades to regulate a variety of cellular processes, including proliferation, migration, differentiation, and cell death. Contrary to a widely held opinion, we show here that nucleoside 5′-O-monophosphorothioate analogs, containing a sulfur atom in a place of one nonbridging oxygen atom in a phosphate group, act as ligands for selected P2Y subtypes. We pay particular attention to the unique activity of thymidine 5′-O-monophosphorothioate (TMPS) which acts as a specific partial agonist of the P2Y6 receptor (P2Y6R). We also collected evidence for the involvement of the P2Y6 receptor in human epithelial adenocarcinoma cell line (HeLa) cell migration induced by thymidine 5′-O-  monophosphorothioate analog. The stimulatory effect of TMPS was abolished by siRNA-mediated P2Y6 knockdown and diisothiocyanate derivative MRS 2578, a selective antagonist of the P2Y6R. Our results indicate for the first time that increased stability of thymidine 5′-O-monophosphorothioate as well as its affinity toward the P2Y6R may be responsible for some long-term effects mediated by this receptor.

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Cloning, Expression, Purification, Regulation, and Subcellular Localization of a Mini-protein from Campylobacter jejuni

Significance Statement

Campylobacter is among the leading causes of bacterial gastroenteritis worldwide. The related disease named campylobacteriosis, affects about 200,000 humans in the European Union each year, which costs about EUR 2.4 billion to public health systems. Despite its impact on human health, this bacteria remains poorly studied and its proteome has not been fully characterized.

The goal of our study was to characterize Cj1169c, a mini-protein from Campylobacter that have never been studied. For this purpose, we cloned the gene Cj1169c in an expression vector in Escherichia coli. Next, we purified the protein which served to produce Cj1169c-specific antibodies that have been used to detect the protein.

Our study has revealed for the first time that Cj1169c is really produced in Campylobacter. We studied the prevalence of this protein in several species of Campylobacter. We found that this protein is only produced in C. jejuni and in C. lari which preferentially colonize birds but not in C. coli (colonizes preferentially pigs). Then, we identified the best growing conditions for Cj1169c protein production. Besides, we demonstrated by several methods that this protein is located in the periplasmic compartment of the bacteria, and could establish via its two cysteines intra- or inter- chain disulfide bridges and may interact with one or more putative partners. According to these data, the Cj1169c protein may be an essential determinant for host colonization.

About The Author

Soumeya Aliouane is a Ph.D student, in Human Pathology; Vascular Pathology and Nutrition at the UMR-MD1, school of medicine, Aix Marseille University, Marseille, France.

About The Author

Dr. Jean-Marie Pagès defended his Doctorat Es-Sciences in 1983. He is Research Director at the French National Institute of Health. JM Pagès has chaired the COST Action BM0701 “Antibiotic transport and efflux” (2008-2012). Heading the UMR_MD1 in Aix-Marseille University, he actively participates in IMI-ND4BB “Molecular basis of the bacterial cell wall permeability” and ITN-Marie Sklodowska-Curie, “Translocation”. He is Member of the SAC of Synchrotron Soleil (Gif/Yvette, France), of the SAC of Instituto de Higiene e Medicina Tropical (Lisboa, Portugal), expert for NSF-USA, NIH-USA, NSERC-Canada, NMRC-Singapour, 7^th PCRDT, etc. He has published over 300 scientific papers, abstracts, patents, and presented lectures in bacteriology-biochemistry-chemistry field.

About The Author

Dr. Jean-Michel Bolla received his Ph.D degree in 1990 from Aix-Marseille University in France, working on the assembly of porins in E. coli. Then, he worked at the Necker Faculty of Medicine in Paris-France, for 2 years as a postdoctoral fellow and studied the iron transport by Listeria monocytogenes. He obtained a permanent position in 1992 at the French National Institute of Health as a research assistant. JM Bolla works on Campylobacter since more than 20 years. His main research interest is the envelope of Gram Negative Bacteria, and he recently focused his activity on antibiotic transport through bacterial envelope. He is now head of the team « New Molecules and new targets » of the UMR-MD1.

Journal Reference

Curr Microbiol. 2016 Jan 11.

Soumeya Aliouane, Jean-Marie Pagès, and Jean-Michel Bolla

Aix-Marseille Université, IRBA, TMCD2 UMR-MD1, Faculté de Médecine, 13385 Marseille, France.

e-mail: jean-michel.bolla@univ-amu.fr

Abstract: 

The Cj1169c-encoded putative protein of Campylobacter was expressed and purified from E. coli after sequence optimization. The purified protein allowed production of a specific rabbit antiserum that was used to study the protein expression in vitro and its subcellular localization in the bacterial cell and putative interactions with other proteins. This protein is produced in Campylobacter and it clearly localizes into the periplasmic space. The level of protein production depends on factors, including pH, temperature, osmolarity and growth phase suggesting a role in the Campylobacter environmental adaptation. The cysteine residues present in the sequence are probably involved in disulfide bridges, which may promote covalent interactions with other proteins of the Campylobacter envelope..

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Scanning Nanospin Ensemble Microscope for Nanoscale Magnetic and Thermal Imaging

Significance Statement

Scanning probe microscopy techniques provide invaluable access to the nanoworld, enabling measurement of local topographic, electronic, optical and magnetic properties depending on the probe used. The emergence of spin-based quantum probes opens up new imaging modalities that can be combined in a single instrument. Here we introduce a “scanning nano-spin ensemble microscope”, in which the microscope probe consists of a small ensemble of optically active quantum spins hosted in a diamond nanocrystal. We illustrate the practicality and versatility of this approach by performing nanoscale imaging of static and fluctuating magnetic fields as well as thermal imaging, at room temperature with typical acquisition times of an hour per image. This opens up new opportunities for nanoscale investigation of a wide variety of materials and phenomena, including in biological settings.

About The Author

Jean-Philippe Tetienne was born in France in 1987. He received his master’s degree in electrical engineering from the Ecole Normale Superieure de Cachan, France, in 2011. During his master’s, he carried out two research projects in the field of nanophotonics, with Dr Raffaele Colombelli at the University of Paris-Sud, and then with Prof Federico Capasso at Harvard University. In 2014, he received his PhD in physics from the Ecole Normale Superieure de Cachan, where, under the supervision of Dr Vincent Jacques, he developed a new type of scanning probe microscope based on a single spin quantum sensor. In particular, he used this microscope to study domain walls in ultrathin ferromagnetic wires, which has led to the discovery of light-assisted domain wall dragging. In 2015, he joined the group of Prof Lloyd Hollenberg at the University of Melbourne, where he is currently working as a postdoctoral fellow on sensing and imaging using diamond quantum sensors. His research interests include nanophotonics, quantum sensing, scanning probe microscopy, and nanomagnetism.

Journal Reference

Nano Lett. 2016 Jan 13;16(1):326-33.

Tetienne JP^1,2, Lombard A^1,3, Simpson DA⁴, Ritchie C², Lu J², Mulvaney P², Hollenberg LC^1,2,4

Show Affiliations

1. Centre for Quantum Computation and Communication Technology, The University of Melbourne , Melbourne Victoria 3010, Australia.
2. Bio21 Institute and School of Chemistry, The University of Melbourne , Melbourne Victoria 3010, Australia.
3. Département de Physique, Ecole Normale Supérieure de Cachan , 94235 Cachan, France.
4. School of Physics, The University of Melbourne , Melbourne Victoria 3010, Australia.

Abstract

Quantum sensors based on solid-state spins provide tremendous opportunities in a wide range of fields from basic physics and chemistry to biomedical imaging. However, integrating them into a scanning probe microscope to enable practical, nanoscale quantum imaging is a highly challenging task. Recently, the use of single spins in diamond in conjunction with atomic force microscopy techniques has allowed significant progress toward this goal, but generalization of this approach has so far been impeded by long acquisition times or by the absence of simultaneous topographic information. Here, we report on a scanning quantum probe microscope which solves both issues by employing a nanospin ensemble hosted in a nanodiamond. This approach provides up to an order of magnitude gain in acquisition time while preserving sub-100 nm spatial resolution both for the quantum sensor and topographic images. We demonstrate two applications of this microscope. We first image nanoscale clusters of maghemite particles through both spin resonance spectroscopy and spin relaxometry, under ambient conditions. Our images reveal fast magnetic field fluctuations in addition to a static component, indicating the presence of both superparamagnetic and ferromagnetic particles. We next demonstrate a new imaging modality where the nanospin ensemble is used as a thermometer. We use this technique to map the photo induced heating generated by laser irradiation of a single gold nanoparticle in a fluid environment. This work paves the way toward new applications of quantum probe microscopy such as thermal/magnetic imaging of operating microelectronic devices and magnetic detection of ion channels in cell membranes.

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A liposomal method for evaluation of inhibitors of H(+)-coupled multidrug transporters

Journal Reference

J Pharmacol Toxicol Methods. 2016 Jan-Feb;77:53-7.

Melchior DL¹, Brill S², Wright GE³, Schuldiner S².

Show Affiliations

1. GLSynthesis Inc., One Innovation Drive, Worcester, MA 01605, USA. Electronic address: donald.melchior@glsynthesis.com.
2. Department of Biological Chemistry, Alexander Silberman Institute of Life Sciences, Edmond J. Safra Campus, Hebrew University, Jerusalem 91904, Israel.
3. GLSynthesis Inc., One Innovation Drive, Worcester, MA 01605, USA.

Abstract

INTRODUCTION:

This paper describes a novel technique, Fluorosomes, applied to investigating the interaction of antimicrobials with proton driven microbial efflux transporters. These transporters remove toxic compounds from the cytoplasm, including antibiotics and are involved in antibiotic resistance.

METHODS:

To assess transporter activity we developed a methodology to generate a proton gradient across Fluorosome membranes into which selected purified fully active efflux transporters were reconstituted. The interior of the Fluorosome particle (a unilamellar liposome) contains a fluorescent drug sensing probe whose fluorescence is quantitatively quenched by transporter substrates. Using an injecting fluorescence plate reader to initiate a proton gradient and to monitor subsequent fluorescence change, real time transport kinetics can be followed and transport inhibition characterized.

RESULTS:

Fluorosomes containing the Escherichia coli EmrE efflux pump demonstrated transport of two known EmrE substrates, ethidium and methyl viologen upon creation of a proton gradient. For Fluorosomes containing the inactive EmrE mutant, E14Q, no transport was observed. When the gradient was fully collapsed by the addition of nigericin, full inhibition of substrate transport was observed. The IC50 for nigericin inhibition of ethidium was shown to be 0.71μM.

DISCUSSION:

We have for the first time prepared and validated a single bacterial efflux pump assay, Fluorosome-trans-EmrE, that faithfully mimics properties of the transporter in vivo. It is faster than whole cell screens, simple to use, amenable to robotics, and reports on very specific targets. We have demonstrated proof of principle with EmrE and have created the first of an intended series of proton driven Fluorosomes.

Copyright © 2015 Elsevier Inc. All rights reserved.

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Involvement of the Tyro3 receptor and its intracellular partner Fyn signaling in Schwann cell myelination

Figure Legend: Mitochondria harboring spastic paraplegia (SPG) 13-associated mutations are co-localized with cleaved (active) caspase 3. Two amino acid mutations Val-98-to-Ile (V98I, upper panels) and Gln-461-to-Glu (Q461E, lower panels) of mitochondrial heat shock protein HSPD1 are associated with SPG13, triggering an apoptotic molecular cascade. Green and red colors indicate mutant HSPD1 and cleaved caspase 3.

About The Author

 Junji Yamauchi

Affiliation: Group Leader, Molecular Pharmacology Group, Department of Pharmacology, National Research Institute for Child Health and Development, Tokyo, Japan.

Research interest: Exploring new molecules underlying myelination and demyelination in the PNS and CNS.

Journal Reference

Mol Biol Cell. 2015 Oct 1;26(19):3489-503.

Miyamoto Y¹, Torii T¹, Takada S², Ohno N³, Saitoh Y³, Nakamura K¹, Ito A⁴, Ogata T⁵, Terada N⁶, Tanoue A¹, Yamauchi J⁷.

Show Affiliations

1. Department of Pharmacology, National Research Institute for Child Health and Development, Setagaya, Tokyo 157-8535, Japan.
2. Department of Systems BioMedicine, National Research Institute for Child Health and Development, Setagaya, Tokyo 157-8535, Japan.
3. Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Chuo, Yamanashi 409-3898, Japan.
4. Research Center, Nissei Bilis, Koga, Shiga 528-0052, Japan.
5. Department of Rehabilitation for the Movement Functions, National Rehabilitation Center for Persons with Disabilities Research Institute, Tokorozawa, Saitama 359-8555, Japan.
6. Graduate School of Medicine, Shinshu University, Matsumoto, Nagano 390-8621, Japan.
7. Department of Pharmacology, National Research Institute for Child Health and Development, Setagaya, Tokyo 157-8535, Japan Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Bunkyo, Tokyo 113-8510, Japan. Email:  yamauchi-j@ncchd.go.jp).

Abstract

During early development of the peripheral nervous system, Schwann cell precursors proliferate, migrate, and differentiate into premyelinating Schwann cells. After birth, Schwann cells envelop neuronal axons with myelin sheaths. Although some molecular mechanisms underlying myelination by Schwann cells have been identified, the whole picture remains unclear. Here we show that signaling through Tyro3 receptor tyrosine kinase and its binding partner, Fyn nonreceptor cytoplasmic tyrosine kinase, is involved in myelination by Schwann cells. Impaired formation of myelin segments is observed in Schwann cell neuronal cultures established from Tyro3-knockout mouse dorsal root ganglia (DRG). Indeed, Tyro3-knockout mice exhibit reduced myelin thickness. By affinity chromatography, Fyn was identified as the binding partner of the Tyro3 intracellular domain, and activity of Fyn is down-regulated in Tyro3-knockout mice, suggesting that Tyro3, acting through Fyn, regulates myelination. Ablating Fyn in mice results in reduced myelin thickness. Decreased myelin formation is observed in cultures established from Fyn-knockout mouse DRG. Furthermore, decreased kinase activity levels and altered expression of myelination-associated transcription factors are observed in these knockout mice. These results suggest the involvement of Tyro3 receptor and its binding partner Fyn in Schwann cell myelination. This constitutes a newly recognized receptor-linked signaling mechanism that can control Schwann cell myelination.

© 2015 Miyamoto et al. This article is distributed by The American Society for Cell Biology under license from the author(s).

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Soft Graphene Nanofibers Designed for the Acceleration of Nerve Growth and Development

Significance Statement

Understanding in the cells responses to programmed electrical signals is a vital tool for the study of the various biological research fields from bioelectronics to regenerative medicine, especially for the nerve engineering. In this study, a novel soft graphene nanofibers was successfully prepared by controlling the assembly of graphene oxide sheets on the surface of electrospun polymer nanofibers, and followed by chemical reduction with hydriodic acid. The graphene nanofibers have ultrathin graphene shells that wrapped the entire surface of the polymer nanofiber, which led to a flexible feature and recoverable electrical conductivity, and physicochemical stability in aqueous solutions with pH values from 2.0 to 11.0. By using the graphene nanofibers for cellular electrical stimulation, a sustained and irreversible change on the electron charge density and the location of the opened Ca²⁺ channel in the cell membrane was achieved, which avoided the common inhibitory effect of the long-term electrical stimulation on the replenishment of Ca²⁺ store, and resulted in a fast and sustainable promotion on the neurons growth and development. The speed of the neurites elongation and maturation of these neurons was almost two times than that of the neurons on the tissue culture plates and graphene film with or without electrical stimulation. We envision that the graphene nanofibers maybe can serve asa powerful tool for developing future therapies for nerve-related disease and injuries in central and peripheral nervous system. In addition, the successful formation of graphene shells in the complex 3D nanofibers extended the superior properties of graphene from traditional 2D nanometer scales to 3D micrometer scales by controlled assembly of graphene oxide sheets in 3D architectural geometry. We expect that this novel preparation technique will open a new way to adapt the graphene macroscopic real-world applications, not only to biomedicine, but also to chemical, electronic, energy, and environmental applications. 

 

About The Author

Zhang-Qi Feng completed a visiting scholarship at The University of Michigan in Ann Arbor as an exchange student (2008–2010), and then earned his Ph.D. (2010) degree in Biomedical Engineering from Southeast University. In fall 2010, he moved to Sun Yat-Sen University as a Lecturer supported by the “100 Talents Program” in the Department of Biomedical Engineering. Since spring 2014, he held an academic position in the School of Chemical Engineering of Nanjing University of Science & Technology. Feng developed several new micro/nano fibrous scaffolds by electrospinning, and has pioneered the application of electrospun fibers to bioartificial liver. His current research interests include the development of bio-inspired materials to improve tissue regeneration and works on advancing understanding the fundamental interaction between biomaterials and biological systems.

 

About The Author

Ting Wang completed a visiting scholarship at The University of Michigan in Ann Arbor as an exchange student (2008–2010), and then received her Ph.D. (2010) degree in Biomedical Engineering from Southeast University. In spring 2011, Ting got the position as a lecturer in the State Key Laboratory of Bioelectronics in the Southeast University. Ting current devotes her attention to developed several new micro/nanoparticles for drug delivery and its bio-safety, and understanding the fundamental interaction between nanoparticle and cell membrane.

 

About The Author

Jia-Cheng Li is currently carrying out master’s degree research with Prof. Zhang-Qi Feng at Nanjing University of Science & Technology. He received his Bachelor of Engineering in Pharmaceutical engineering from the University of Huang Shan, Anhui province, China in 2014. His research interests include the synthesis, properties, and applications of multiphase micro/nanoparticles.

 

About The Author

Bin Zhao is currently pursuing his master’s degree in Materials Science at Nanjing University of Science and Technology with Prof. Zhang-Qi Feng. He received a bachelor degree (2010) in Materials Chemistry from Binzhou University of China. His current research interests focus on creating short electrospun functional fibers for modulating the behavior of stem cells and on understanding the cell signal transduction in short fibers dispersions.

Journal Reference

Adv Mater. 2015 Nov;27(41):6462-8.

Feng ZQ¹, Wang T², Zhao B¹, Li J¹, Jin L³.

Show Affiliations

1. School of Chemical Engineering, Nanjing University of Science and Technology, 200 Xiao Ling Wei, Nanjing, 210094, China.
2. State Key Laboratory of Bioelectronics, Southeast University, Nanjing, 210096, China.
3. School of Engineering, Sun Yat-Sen University, 51006, China.

Abstract

Soft graphene nanofibers with recoverable electrical conductivity and excellent physicochemical stability are prepared by a controlled assembly technique. By using the soft graphene nanofibers for cellular electrical stimulation, the common inhibitory effect of long-term electrical stimulation onnerve growth and development is avoided, which usually happens with traditional 2D conductive materials.

© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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Notch signaling represses GATA4-induced expression of genes involved in steroid biosynthesis

Significance Statement

The Notch signaling pathway regulates cell fate decisions in many tissues.  We, and others, have demonstrated that Notch signaling pathway genes are expressed in a dynamic fashion in granulosa cells of developing follicles, and in Leydig and Sertoli cells of the adult testis. However, the downstream target genes of Notch-signaling had remained elusive. In this study, we provide the first evidence that Notch signaling inhibits the expression of genes in the steroid biosynthetic pathway. The promoters of specific enzymes involved in steroid biosynthesis, Star, HSD3B2, and Cyp19aI, were repressed by activated Notch, in the presence of their activator, GATA4. We postulate that Notch signaling may mediate the biphasic response of granulosa cells to paracrine cues such as FSH. In growing preantral follicles active Notch signaling represses expression of steroid synthetic genes, thus inducing a proliferative response, but in larger follicles, Notch signaling is attenuated and FSH induces differentiation. The Notch signaling pathway genes are expressed in the pituitary and adrenal glands as well. These data indicate that Notch signaling could have a global effect on steroid synthesis within the hypothalamic-pituitary-gonadal axis; this will be an important future consideration.

About The Author

Dr. Jeanne Wilson-Rawls is the corresponding author on this study. She received a PhD in Cellular and Molecular Biology from St. Louis School of Medicine and completed a post-doctoral fellowship with Dr. Eric N. Olson at UT Southwestern Medical Center at Dallas. Currently, she is an associate professor of Genomics, Evolution & Bioinformatics in the School of Life Sciences at Arizona State University.  Her research group has been studying Notch signaling and cell lineage decisions in the development and regeneration of muscle and development and maturation of germ cells. 

 

 

Journal Reference

Reproduction. 2015;150(4):383-94.

George RM¹, Hahn KL¹, Rawls A¹, Viger RS^2,3, Wilson-Rawls J¹.

Show Affiliations

1. School of Life Sciences,Arizona State University, PO Box 874501, Tempe, Arizona 85827-45012, USA
2. Reproduction,Mother and Child Health, Centre de Recherche du CHU de Québec and Centre de Recherche en Biologie de la Reproduction (CRBR), Quebec City, Quebec, Canada
3. Department of Obstetrics,Gynecology, and Reproduction, Laval University, Quebec City, Quebec, Canada G1K 7P4

Abstract

Notch2 and Notch3 and genes of the Notch signaling network are dynamically expressed in developing follicles, where they are essential for granulosa cell proliferation and meiotic maturation. Notch receptors, ligands, and downstream effector genes are also expressed in testicular Leydig cells, predicting a potential role in regulating steroidogenesis. In this study, we sought to determine if Notch signaling in small follicles regulates the proliferation response of granulosa cells to FSH and represses the up-regulation steroidogenic gene expression that occurs in response to FSH as the follicle grows. Inhibition of Notch signaling in small preantral follicles led to the up-regulation of the expression of genes in the steroid biosynthetic pathway. Similarly, progesterone secretion by MA-10 Leydig cells was significantly inhibited by constitutively active Notch. Together, these data indicated that Notch signaling inhibits steroidogenesis. GATA4 has been shown to be a positive regulator of steroidogenic genes, including STAR protein, P450 aromatase, and 3B-hydroxysteroid dehydrogenase. We observed that Notch downstream effectors HEY1, HEY2, and HEYL are able to differentially regulate these GATA4-dependent promoters. These data are supported by the presence of HEY/HES binding sites in these promoters. These studies indicate that Notch signaling has a role in the complex regulation of the steroidogenic pathway.

© 2015 Society for Reproduction and Fertility.

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Vitronectin promotes the progress of the initial differentiation stage in cerebellar granule cells

Journal Reference

Mol Cell Neurosci. 2016 Jan;70:76-85.

Hashimoto K¹, Sakane F¹, Ikeda N¹, Akiyama A¹, Sugahara M¹, Miyamoto Y².

Show Affiliations

1. Graduate School of Humanities and Sciences, Ochanomizu University, Otsuka 2-1-1, Bunkyo-ku, Tokyo 112-8610, Japan.
2. Graduate School of Humanities and Sciences, Ochanomizu University, Otsuka 2-1-1, Bunkyo-ku, Tokyo 112-8610, Japan. Electronic address: miyamoto.yasunori@ocha.ac.jp.

Abstract

Vitronectin (VN), which is an extracellular matrix protein, is known to be involved in the proliferation and differentiation of primary cultured cerebellar granule cell precursors (CGCPs); however, the effect of Vitronectin is not fully understood. In this study, we analyzed the effects of Vitronectin loss on the proliferation and differentiation of CGCPs in Vitronectin knockout (VNKO) mice in vivo. First, immunohistochemistry showed that Vitronectin was distributed in the region from the inner external granule layer (iEGL) through the internal granule layer (IGL) in wild-type (WT) mice. Next, we observed the formation of the cerebellar cortex using sagittal sections of VNKO mice at postnatal days (P) 5, 8 and 11. Loss of Vitronectin suppressed the ratio of NeuN, a neuronal differentiation marker, to positive   cerebellar granule cells (CGCs) in the external granule layer (EGL) and the ratio of CGCs in the IGL at P8, indicating that the loss of Vitronectin suppresses the differentiation into CGCs. However, the loss of Vitronectin did not significantly affect the proliferation of CGCPs. Next, the effect of Vitronectin loss on the initial differentiation stage of CGCPs was examined. The loss of Vitronectin increased the expression levels of Transient axonal glycoprotein 1 (TAG1), a marker of neurons in the initial differentiation stage, in the cerebella of VNKO mice at P5 and 8 and increased the ratio of TAG1-positive cells in the primary culture of VNKO-derived CGCPs, indicating that the loss of VN accumulates the CGCPs in the initial differentiation stage. Taken together, these results demonstrate that Vitronectin promotes the progress of the initial differentiation stage of CGCPs.

Copyright © 2015 Elsevier Inc. All rights reserved.

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Hyperphosphatemia, Phosphoprotein Phosphatases, and Microparticle Release in Vascular Endothelial Cells

Significance Statement

Cardiovascular disease is the leading cause of death in patients with chronic kidney disease. More than half of the patients with kidney diseases develop vascular complications at some stage of their disease progression. Hyperphosphatemia, high serum inorganic phosphate (Pi) levels, is a common biochemical abnormality in kidney patients and can exert damaging effects on endothelial cells lining the blood vessels. Microparticles, small 100-1000nm in diameter cell membrane fragments, are released from cells under stress. While hyperphosphatemia can exert its damaging effect on cells of the vasculature by inducing soft-tissue calcification, we have proposed that Pi can exert functional effects on the cells of the blood vessels (especially endothelial cells), resulting in generation of MPs by a direct inhibition of phosphoprotein phosphatases resulting in cytoskeletal protein dysregulation and subsequent MP formation. The mechanism of this Pi-induced cell stress and microparticle formation is elusive. In this study we have shown a novel mechanism by which Pi induces cell-stress, resulting in the generation of pro-coagulant forms of endothelial MPs which may contribute to acute occlusive events. The major finding of our study is that inorganic phosphate concentrations similar to those observed in hyperphosphatemia in kidney patients trigger an acute release of pro-coagulant microparticles from human endothelial cells. This is of direct interest to nephrologists because it provides a molecular basis for the observed link between hyperphosphatemia and cardiovascular disease in kidney patients. It is also of interest to a wider audience because the proposed mechanism provides a widely applicable explanation for pathological effects of phosphate excess in mammalian cells. Our study provides a novel pathologic link between hyperphosphatemia, generation of MPs, and thrombotic risk.

About The Author

Nima Abbasian, Ph.D.

BSc in Biology (Microbiology) Azad University, Iran (2006)

MSc in Infection & Immunity, University of Leicester, UK (2010)

Ph.D. in Infection, Immunity, & Inflammation, University of Leicester, UK (2015)

KRUK Postdoctoral Research Fellow, University of Leicester, UK (2015-Present)

Research interests

My research focuses on cellular and molecular mechanisms of inorganic phosphate (Pi) induced stress on blood cells and endothelial cell dysfunction in kidney disease. I am particularly interested in: a) The regulation of intracellular concentration of inorganic phosphate (Pi) by plasma membrane solute transporter proteins in human endothelial cells in response to higher extracellular Pi concentration (known as hyperphosphatemia) and subsequent signal transductions induced by Pi in the cells, and b) The mechanism of high Pi-induced stress in cells and subsequent release of cell membrane-derived microparticle (MPs). I have focused particularly on the mechanism of inorganic phosphate signalling to cytoskeletal proteins and subsequent clinically important pro-coagulant endothelial MP release that acutely starts budding of the cell membrane during uremic cardiovascular disease.

Journal Reference

J Am Soc Nephrol. 2015;26(9):2152-62.

Abbasian N¹, Burton JO², Herbert KE³, Tregunna BE¹, Brown JR², Ghaderi-Najafabadi M³, Brunskill NJ², Goodall AH³, Bevington A⁴.

Show Affiliations

1. Departments of Infection, Immunity and Inflammation and.
2. Departments of Infection, Immunity and Inflammation and John Walls Renal Unit, University Hospitals of Leicester, Leicester, United Kingdom; and.
3. Cardiovascular Sciences, University of Leicester, Leicester, United Kingdom; Leicester National Institute for Health Research Cardiovascular Biomedical Research Unit Cardiovascular Sciences, University of Leicester, United Kingdom.
4. Departments of Infection, Immunity and Inflammation and ab74@leicester.ac.uk.

Abstract

Hyperphosphatemia in patients with advanced CKD is thought to be an important contributor to cardiovascular risk, in part because of endothelial cell (EC) dysfunction induced by inorganic phosphate (Pi). Such patients also have an elevated circulating concentration of procoagulant endothelial microparticles (MPs), leading to a prothrombotic state, which may contribute to acute occlusive events. We hypothesized that hyperphosphatemialeads to MP formation from ECs through an elevation of intracellular Pi concentration, which directly inhibits  phosphoprotein  phosphatases, triggering a global increase in phosphorylation and cytoskeletal changes. In cultured human ECs (EAhy926), incubation with elevated extracellular Pi (2.5 mM) led to a rise in intracellular Pi concentration within 90 minutes. This was mediated by PiT1/slc20a1 Pi transporters and led to global accumulation of tyrosine- and serine/threonine-phosphorylated proteins, a marked increase in cellular Tropomyosin-3, plasma membrane blebbing, and release of 0.1- to 1-μm-diameter MPs. The effect of Pi was independent of oxidative stress or apoptosis. Similarly, global inhibition of phosphoprotein phosphatases with orthovanadate or fluoride yielded a global protein phosphorylation response and rapid release of MPs. The Pi-induced MPs expressed VE-cadherin and superficial phosphatidylserine, and in a thrombin generation assay, they displayed significantly more procoagulant activity than particles derived from cells incubated in medium with a physiologic level of Pi (1 mM). These data show a mechanism of Pi-induced cellular stress and signaling, which may be widely applicable in mammalian cells, and in ECs, it provides a novel pathologic link between hyperphosphatemia, generation of MPs, and thrombotic risk.

Copyright © 2015 by the American Society of Nephrology.

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Role of high-mobility group box 1 in methamphetamine-induced activation and migration of astrocytes

About The Author

Dr.  Honghong Yao, Professor at the Southeast University, Department of Pharmacology. Dr. Yao considered to be an international expert in the field of neuroscience.  As such, she has published extensively in academic, peer-reviewed journals and am a frequent invitee and speaker at international conferences and various research institutions and universities within the US, Europe, and other parts of the world.  In addition, she has received a $3 million Medical Research Council group grant; have served on numerous national and international committees; and have received numerous scholastic honors and awards.  

Journal Reference

J Neuroinflammation. 2015;12:156.

Zhang Y¹, Zhu T², Zhang X¹, Chao J³, Hu G⁴, Yao H^5,6.

Show Affiliations

1. Department of Pharmacology, Medical School of Southeast University, Nanjing, 210009, China.
2. Department of Cardiology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu, 210029, China.
3. Department of Physiology, Medical School of Southeast University, Nanjing, China.
4. Department of Pharmacology, Nanjing Medical University, Nanjing, China.
5. Department of Pharmacology, Medical School of Southeast University, Nanjing, 210009, China. yaohh@seu.edu.cn.
6. Institute of Life Sciences, Key Laboratory of Developmental Genes and Human Disease, Southeast University, Nanjing, Jiangsu, China. yaohh@seu.edu.cn.

Abstract

BACKGROUND:

Mounting evidence has indicated that high-mobility group box 1 (HMGB1) is involved in cell activation and migration. Our previous study demonstrated that methamphetamine mediates    activation of astrocytes via sigma-1 receptor (σ-1R). However, the elements downstream of σ-1R in this process remain poorly understood. Thus, we examined the molecular mechanisms involved in  astrocyte  activation and migration induced by methamphetamine.

METHODS:

The expression of HMGB1, σ-1R, and glial fibrillary acidic protein (GFAP) was examined by western blot and immunofluorescent staining. The phosphorylation of cell signaling pathways was detected by western blot, and cell migration was examined using a wound-healing assay in rat C6 astroglia-like cells transfected with lentivirus containing red fluorescent protein (LV-RFP) as well as in primary human astrocytes. The role of HMGB1 in astrocyte activation and migration was validated using a siRNA approach.

RESULTS:

Exposure of C6 cells to methamphetamine increased the expression of HMGB1 via the activation of σ-1R, Src, ERK mitogen-activated protein kinase, and downstream NF-κB p65 pathways. Moreover, methamphetamine treatment resulted in increased cell activation and migration in C6 cells and primary human astrocytes. Knockdown of HMGB1 in astrocytes transfected with HMGB1 siRNA attenuated the increased cell activation and migration induced by methamphetamine, thereby implicating the role of HMGB1 in the activation and migration of C6 cells and primary human astrocytes.

CONCLUSIONS:

This study demonstrated that methamphetamine-mediated activation and migration of astrocytes involved HMGB1 up-regulation through an autocrine mechanism. Targeting HMGB1 could provide insights into the development of a potential therapeutic approach for alleviation of cell activation and migration of astrocytes induced by methamphetamine.

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Serum α-klotho concentrations during preimplantation can predict aging or quality of human oocytes and clinical pregnancy rates

Significance Statement

 Many researches in the world are very interested in the aging or quality of human oocytes. However, a simple biomarker to predict the aging or quality of human oocytes is not discovered. On the other hand, α-klotho is known as an anti-aging molecule since 1997 (Kuro-o M, et al., Nature). Therefore, increases in α-klotho concentrations in human serum positively may improve the aging and quality of human oocytes.

The present research (a prospective cohort study) is the first study reporting that the serum α-klotho concentrations during preimplantation can predict the aging or quality of human oocytes and clinical pregnancy rates.

Furthermore, the present research suggests that the serum α-klotho concentrations during preimplantation may be improved by vitamin D supplementation. Therefore, the aging or quality of human oocytes may be improved by vitamin D supplementation, although clinical trials will be needed in the near future.  In conclusion, the present research is a breakthrough study in reproductive medicine.

Journal Reference

Springerplus. 2016 Jan 20;5:53.

Takemura T, Okabe M.

Reproductive Medicine Institute Japan, Nakano-ku, Chuo, 3-37-12, Tokyo, 164-0011 Japan.

Abstract

BACKGROUND:

To discover simple biomarkers to evaluate the aging or quality of human oocytes and clinical pregnancy rates is needed. However, the association among serum α-klotho concentrations during preimplantation, the aging  or quality of human oocytes and clinical pregnancy rates has not been investigated.

FINDINGS:

The serum α-klotho concentrations during preimplantation decreased due to aging (p < 0.001), whereas the maturation rates of human oocytes (p < 0.001) and the fertilization rates (p < 0.001) improved in association with increased serum α-klotho concentrations. Furthermore, multiple logistic regression analysis showed that the clinical pregnancy rates were influenced by serum α-klotho concentrations during preimplantation (p < 0.001), the patient’s age (p = 0.003), maturation rates of  human oocytes (p < 0.001), fertilization rates (p < 0.001) and the serum 25 (OH) D levels (p < 0.001) regardless of race (p = 0.29) and BMI (p = 0.96).

CONCLUSION:

The serum α-klotho concentrations during preimplantation would be a simple biomarker in order to  predict the aging or quality of human oocytes and clinical pregnancy rates.

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The membrane-associated MUC1 improves adhesion of salivary MUC5B on buccal cells. Application to development of an in vitro cellular model of oral epithelium

Significance Statement

The mucosal pellicle is a thin layer of salivary proteins lining epithelial oral cells. This pellicle has a protective function of oral mucosae against abrasion, pathogenic microorganisms or chemical xenobiotics. However, the currently available cellular models of oral mucosa do not consider this layer. We therefore developed a model based on TR146 cells, stably transfected in order to express the membrane-associated mucin MUC1. From a morphological point of view, the cells exhibited the appearance of non-keratinized oral mucosal cells, with typical membrane folds. The transfected cells could retain a higher quantity of the salivary mucins MUC5B than the native cells, highlighting that the membrane-associated mucins favour binding of salivary mucins to epithelial oral cells. We also showed that confluent transfected cells incubated with human saliva were lined with a “pellicle”, i.e. a thin filamentous salivary network. This in vitro model is a promising tool to investigate biological events occurring at the surface of oral mucosae, such as permeability to drugs or microorganism invasion. It may also be useful to study molecular interactions between food constituents and mucosae at the origin of some sensory properties of food products (astringency, aroma persistence…). 

 

Journal Reference

Arch Oral Biol. 2016 Jan;61:149-55.

Ployon S¹, Belloir C¹, Bonnotte A², Lherminier J³, Canon F¹, Morzel M⁴.

Show Affiliations

1. CNRS, UMR 6265 Centre des Sciences du GoÛt et de l’Alimentation, F-21000 Dijon, France; INRA, UMR1324 Centre des Sciences du GoÛt et de l’Alimentation, F-21000 Dijon, France; Université de Bourgogne, UMR Centre des Sciences du GoÛt et de l’Alimentation, F-21000 Dijon, France.
2. Université de Bourgogne, UMR 1347 Agroécologie, Plateforme DImaCell, Centre de Microscopie INRA/Université de Bourgogne, F-21000 Dijon, France.
3. INRA, UMR1347 Agroécologie, ERL CNRS 6300, Plateforme DImaCell, Centre de Microscopie INRA/Université de Bourgogne, F-21000 Dijon, France.
4. CNRS, UMR 6265 Centre des Sciences du GoÛt et de l’Alimentation, F-21000 Dijon, France; INRA, UMR1324 Centre des Sciences du GoÛt et de l’Alimentation, F-21000 Dijon, France; Université de Bourgogne, UMR Centre des Sciences du GoÛt et de l’Alimentation, F-21000 Dijon, France. Electronic address: martine.morzel@dijon.inra.fr.

Abstract

OBJECTIVES:

The mucosal pellicle is a thin layer of salivary proteins, mostly MUC5B mucins, anchored to epithelial oral cells. This pellicle is involved in protection of oral mucosae against abrasion, pathogenic microorganisms or chemical xenobiotics. The present study aimed at studying the involvement of MUC1 in mucosal pellicle formation and more specifically in salivary MUC5B binding using a cell-based model of oral epithelium.

DESIGN:

MUC1 mRNAs were not detected in TR146 cells, and therefore a stable cell line named TR146/MUC1 expressing this protein was developed by transfection. TR146 and TR146/MUC1 were incubated with human saliva in order to evaluate retention of MUC5B by epithelial cells.

RESULTS:

The cell surface of both TR146 and TR146/MUC1 was typical of a squamous non-keratinized epithelium, with the presence of numerous microplicae. After incubation for 2h with saliva diluted in culture medium (1:1) and two washes with PBS, saliva deposits on cells appeared as a loose filamentous thin network. MUC5B fluorescent immunostaining evidenced a heterogeneous lining of confluent cell cultures by this salivarymucin but with higher fluorescence on TR146/MUC1 cells. Semi-quantification of MUC5B bound to cells confirmed a better retention by TR146/MUC1, evaluated by Dot Blot (+34.1%, p<0.05) or by immunocytochemistry (+44%, p<0.001).

CONCLUSION:

The membrane-bound mucin MUC1 is a factor enhancing the formation of the mucosal pellicle by increasing the binding of salivaryMUC5B to oral epithelial cells. An in vitro model suitable to study specifically the function and properties of the mucosal pellicle is proposed.

Copyright © 2015 Elsevier Ltd. All rights reserved.

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A new strategy to develop the disposable label-free immunosensor with electrochemiluminescent probing

Journal Reference

Journal of Electroanalytical Chemistry, Volume 747, 2015, Pages 136–142.

Lin Jiang¹, Ya Yang^1,2 , Yifeng Tu^1,3

Show Affiliations

1. Institute of Analytical Chemistry, Soochow University, Dushu Lake Campus of Soochow University, Suzhou Industrial Park, 215123 Suzhou, PR China
2. Department of Forensic Medicine, Soochow University, Dushu Lake Campus of Soochow University, Suzhou Industrial Park, 215123 Suzhou, PR China
3. The Key Lab of Health Chemistry and Molecular Diagnosis of Suzhou, Dushu Lake Campus of Soochow University, Suzhou Industrial Park, 215123 Suzhou, PR China

Abstract

In this paper, a new strategy to prepare the label-free immunosensor signaling with the electrochemiluminescence (ECL) of luminol will be reported. The gold nano-particles (AuNPs) functionalized indium tin oxide coated glass is used as the substrate for antibody immobilization. These AuNPs also improve the ECL behavior of luminol. This strategy for immunosensor construction is simple, fast, cheap, and with no requirements of expensive apparatus. For validating the practicability of this new strategy, with methamphetamine (MA) as target model, an methamphetamine sensor was built by immobilizing the methamphetamine-antibody onto the surface of previously mentioned substrate. The ECL intensity on resultant sensor, after the direct immuno-interaction, decreased proportional to the concentration of methamphetamine in a wide linear range from 2 to 500 ng mL⁻¹ with a detection limit of 0.3 ng mL⁻¹. This proposed sensor is sensitive, specific, stable and reliable. It has been successfully applied to detect the methamphetamine in spiked human serums.

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