bluuebunny/crossref_metadata_2025 huggingface dataset:
Bioinspired Immobilization of Glycerol Dehydrogenase by Metal Ion-Chelated Polyethyleneimines as Artificial Polypeptides
Authors: Zhang Yonghui , Ren Hong , Wang Yali , Chen Kainan , Fang Baishan , Wang Shizhen
Published: April 2016

In this study, a novel, simple and generally applicable strategy for multimeric oxidoreductase immobilization with multi-levels interactions was developed and involved activity and stability enhancements. Linear polyethyleneimines (PEIs) are flexible cationic polymers with molecular weights that span a wide range and are suitable biomimic polypeptides for biocompatible frameworks for enzyme immobilization. Metal ion-chelated linear PEIs were applied as a heterofunctional framework for glycerol dehydrogenase (GDH) immobilization by hydrogen bonds, electrostatic forces and coordination bonds interactions. Nanoparticles with diameters from 250–650 nm were prepared that exhibited a 1.4-fold enhancement catalytic efficiency. Importantly, the half-life of the immobilized GDH was enhanced by 5.6-folds in aqueous phase at 85 °C. A mechanistic illustration of the formation of multi-level interactions in the PEI-metal-GDH complex was proposed based on morphological and functional studies of the immobilized enzyme. This generally applicable strategy offers a potential technique for multimeric enzyme immobilization with the advantages of low cost, easy operation, high activity reservation and high stability.
Mouse Sox17 haploinsufficiency leads to female subfertility due to impaired implantation
Authors: Hirate Yoshikazu , Suzuki Hitomi , Kawasumi Miyuri , Takase Hinako M. , Igarashi Hitomi , Naquet Philippe , Kanai Yoshiakira , Kanai-Azuma Masami
Published: April 2016

Embryonic implantation comprises a dynamic and complicated series of events, which takes place only when the maternal uterine endometrium is in a receptive state. Blastocysts reaching the uterus communicate with the uterine endometrium to implant within a narrow time window. Interplay among various signalling molecules and transcription factors under the control of ovarian hormones is necessary for successful establishment of pregnancy. However, the molecular mechanisms that allow embryonic implantation in the receptive endometrium are still largely unknown. Here, we show that Sry-related HMG box gene-17 (Sox17) heterozygous mutant female mice exhibit subfertility due to implantation failure. Sox17 was expressed in the oviduct, uterine luminal epithelium, and blood vessels. Sox17 heterozygosity caused no appreciable defects in ovulation, fertilisation, blastocyst formation, and gross morphology of the oviduct and uterus. Another group F Sox transcription factor, Sox7, was also expressed in the uterine luminal and glandular epithelium relatively weakly. Despite uterine Sox7 expression, a significant reduction in the number of implantation sites was observed in Sox17 heterozygous mutant females due to haploinsufficiency. Our findings revealed a novel role of Sox17 in uterine receptivity to embryo implantation.
In vivo imaging demonstrates dendritic spine stabilization by SynCAM 1
Authors: Körber Nils , Stein Valentin
Published: April 2016

Formation and stability of synapses are required for proper brain function. While it is well established that synaptic adhesion molecules are important regulators of synapse formation, their specific role during different phases of synapse development remains unclear. To investigate the function of the synaptic cell adhesion molecule SynCAM 1 in the formation, stability and maintenance of spines we used 2-photon in vivo imaging to follow individual spines over a long period of time. In SynCAM 1 knockout mice the survival rate of existing spines was reduced and fewer filopodia-like structures were converted into stable spines. SynCAM 1flag overexpression resulted in more stable spines and fewer filopodia-like structures. When SynCAM 1flag overexpression is turned on the spine density rapidly increases within a few days. Interestingly, the spine density stayed at an elevated level when SynCAM 1flag overexpression was turned off. Our data indicate that the SynCAM 1 induced altered spine density is not caused by the formation of newly emerging protrusions, instead SynCAM 1 stabilizes nascent synaptic contacts which promotes their maturation. Concomitant with the synaptic stabilization, SynCAM 1 generally prolongs the lifetime of spines. In summary, we demonstrate that SynCAM 1 is a key regulator of spine stability.
Robust and tunable itinerant ferromagnetism at the silicon surface of the antiferromagnet GdRh2Si2
Authors: Güttler M. , Generalov A. , Otrokov M. M. , Kummer K. , Kliemt K. , Fedorov A. , Chikina A. , Danzenbächer S. , Schulz S. , Chulkov E. V. , Koroteev Yu. M. , Caroca-Canales N. , Shi M. , Radovic M. , Geibel C. , Laubschat C. , Dudin P. , Kim T. K. , Hoesch M. , Krellner C. , Vyalikh D. V.
Published: April 2016

Spin-polarized two-dimensional electron states (2DESs) at surfaces and interfaces of magnetically active materials attract immense interest because of the idea of exploiting fermion spins rather than charge in next generation electronics. Applying angle-resolved photoelectron spectroscopy, we show that the silicon surface of GdRh2Si2bears two distinct 2DESs, one being a Shockley surface state and the other a Dirac surface resonance. Both are subject to strong exchange interaction with the ordered 4f-moments lying underneath the Si-Rh-Si trilayer. The spin degeneracy of the Shockley state breaks down below ~90 K and the splitting of the resulting subbands saturates upon cooling at values as high as ~185 meV. The spin splitting of the Dirac state becomes clearly visible around ~60 K, reaching a maximum of ~70 meV. An abrupt increase of surface magnetization at around the same temperature suggests that the Dirac state contributes significantly to the magnetic properties at the Si surface. We also show the possibility to tune the properties of 2DESs by depositing alkali metal atoms. The unique temperature-dependent ferromagnetic properties of the Si-terminated surface in GdRh2Si2could be exploited when combined with functional adlayers deposited on top for which novel phenomena related to magnetism can be anticipated.
Chemical characterization and bioactive properties of Geranium molle L.: from the plant to the most active extract and its phytochemicals
Authors: Graça V. C. , Barros Lillian , Calhelha Ricardo C. , Dias Maria Inês , Carvalho Ana Maria , Santos-Buelga Celestino , Ferreira Isabel C. F. R. , Santos P. F.
Published: April 2016

The phytochemical characterization, antioxidant activity and in vitro cytotoxicity against human cancer cell lines of Geranium molle L. extracts are reported for the first time.
Heterogeneous recyclable nano-palladium catalyzed amidation of esters using formamides as amine sources
Authors: Bao Yong-Sheng , Wang Lili , Jia Meilin , Xu Aiju , Agula Bao , Baiyin Menghe , Zhaorigetu Bao
Published: April 2016

Catalyzed by supported palladium nanoparticles, a decarbonylative amidation reaction between various aryl esters and formamides by C–O bond activation has been developed for the synthesis of amides.
Organic charge-transfer complexes for the selective accommodation of aromatic isomers using anthracene derivatives and TCNQ
Authors: Khan Arshad , Wang Mingliang , Usman Rabia , Lu Jiao , Sun Hao , Du Man , Zhang Ruimin , Xu Chunxiang
Published: April 2016

Four organic-charge transfer (CT) complexes (Ct1–Ct4) have been reported. Ct1 discerns p-xylene in the presence of a xylene mixture.
Quantum chemical approach for highly durable anion exchange groups in solid-state alkaline fuel cells
Authors: Matsuyama K. , Ohashi H. , Miyanishi S. , Ushiyama H. , Yamaguchi T.
Published: April 2016

The durability of anion exchange groups against OH− ion that decomposed fast.
Magnetically triggered drug release from biocompatible microcapsules for potential cancer therapeutics
Authors: Bi Hongmei , Ma Shenghua , Li Qingchuan , Han Xiaojun
Published: April 2016

The drug release of two types of MLMs synchronizes the AMF switch; different mechanisms were discussed in this paper.
Determination of parabens in cosmetic products using high performance liquid chromatography with fluorescence detection
Authors: Zgoła-Grześkowiak Agnieszka , Werner Justyna , Jeszka-Skowron Magdalena , Czarczyńska-Goślińska Beata
Published: April 2016

Parabens are preservatives with a broad spectrum of antimicrobial activity widely used in cosmetics, foods and pharmaceuticals.
Layered MoS2–graphene composites for biosensor applications with sensitive electrochemical performance
Authors: Zhang Kunjie , Sun Hong , Hou Shifeng
Published: April 2016

The procedure for the fabrication of layered Mb/MoS2–graphene/Nafion films.
Dye-sensitized photocatalytic hydrogen production: distinct activity in a glucose derivative of a phenothiazine dye
Authors: Manfredi N. , Cecconi B. , Calabrese V. , Minotti A. , Peri F. , Ruffo R. , Monai M. , Romero-Ocaña I. , Montini T. , Fornasiero P. , Abbotto A.
Published: April 2016

A glucose-functionalized phenothiazine dye gave efficient activities compared to the common hydrophilic triethylene glycol derivative as a consequence of improved wettability and peculiar features of the sugar functionality.
Evidence for an intrinsic binding force between dodecaborate dianions and receptors with hydrophobic binding pockets
Authors: Warneke Jonas , Jenne Carsten , Bernarding Johannes , Azov Vladimir A. , Plaumann Markus
Published: April 2016

Gas phase binding studies revealed strong intrinsic intermolecular interactions between dianionic dodecaborates [B12X12]2− and neutral organic hosts.
Thioether bond formation by SPASM domain radical SAM enzymes: Cα H-atom abstraction in subtilosin A biosynthesis
Authors: Benjdia Alhosna , Guillot Alain , Lefranc Benjamin , Vaudry Hubert , Leprince Jérôme , Berteau Olivier
Published: April 2016

The radical SAM enzyme AlbA has been reported to catalyze the formation of a thioether bond in the antibiotic subtilosin A. By modeling, biochemical and synthetic approaches, we propose novel mechanistic perspectives on this emerging group of enzymes.
New two dimensional liquid-phase colorimetric assay based on old iodine–starch complexation for the naked-eye quantitative detection of analytes
Authors: Nie Jinfang , Brown Tom , Zhang Yun
Published: April 2016

This work describes a new point-of-care two dimensional liquid-phase colorimetric assay (2D LPCA) based on old iodine–starch complexation reaction firstly discovered in 1814 for simple, low-cost, portable, naked-eye detection and quantification of two model analytes.
A FRET-enabled molecular peptide beacon with a significant red shift for the ratiometric detection of nucleic acids
Authors: Maity Debabrata , Jiang Juanjuan , Ehlers Martin , Wu Junchen , Schmuck Carsten
Published: April 2016

A cationic molecular peptide beaconNAP1functionalized with a fluorescence resonance energy transfer-pair at its ends allows the ratiometric detection of ds-DNA with a preference for AT rich sequences.
Lower temperature optimum of a smaller, fragmented triphosphorylation ribozyme
Authors: Akoopie Arvin , Müller Ulrich F.
Published: April 2016

The temperature optimum of a triphosphorylation ribozyme shifted to lower temperature, after size reduction and fragmentation.
The unexpectedly rich reconstructions of rutile TiO2(011)-(2 × 1) surface and the driving forces behind their formation: an ab initio evolutionary study
Authors: Wang Qinggao , Oganov Artem R. , Feya Oleg D. , Zhu Qiang , Ma Dongwei
Published: April 2016

In this paper, we employ state-of-the-art theoretical approaches to elucidate the structures of the (011) surface of rutile (R-)TiO2.
A hybrid model of support vector regression with genetic algorithm for forecasting adsorption of malachite green onto multi-walled carbon nanotubes: central composite design optimization
Authors: Ghaedi M. , Dashtian K. , Ghaedi A. M. , Dehghanian N.
Published: April 2016

The aim of this work is the study of the predictive ability of a hybrid model of support vector regression with genetic algorithm optimization (GA–SVR) for the adsorption of malachite green (MG) onto multi-walled carbon nanotubes (MWCNTs).
Graphene and its electrochemistry – an update
Authors: Ambrosi Adriano , Chua Chun Kiang , Latiff Naziah Mohamad , Loo Adeline Huiling , Wong Colin Hong An , Eng Alex Yong Sheng , Bonanni Alessandra , Pumera Martin
Published: April 2016

The electrochemistry of graphene and its derivatives has been extensively researched in recent years. This extends from the electrochemical preparation methods, the electrocatalytic properties of graphene doped with p-block or d-block elements, the capacitive properties to the electrochemical sensing applications.
Hepatoprotective effect of flavonoids from Cirsium japonicum DC on hepatotoxicity in comparison with silymarin
Authors: Ma Qin , Wang Lin-Huan , Jiang Jian-Guo
Published: April 2016

Cirsium japonicum DC is a perennial plant that is widely distributed throughout China.
Glucitol-core containing gallotannins inhibit the formation of advanced glycation end-products mediated by their antioxidant potential
Authors: Ma Hang , Liu Weixi , Frost Leslie , Kirschenbaum Louis J. , Dain Joel A. , Seeram Navindra P.
Published: April 2016

Glucitol-core containing gallotannins (GCGs) are polyphenols containing galloyl groups attached to a 1,5-anhydro-d-glucitol core, which is uncommon among naturally occurring plant gallotannins.
The upcoming 3D-printing revolution in microfluidics
Authors: Bhattacharjee Nirveek , Urrios Arturo , Kang Shawn , Folch Albert
Published: April 2016

A comprehensive exploration of the state-of-the-art and a critical outlook on the advantages, barriers and potential of 3D-printing in microfluidics.
In vitro micro-physiological immune-competent model of the human skin
Authors: Ramadan Qasem , Ting Fiona Chia Wan
Published: April 2016

HacaT/U937 coculture-based in vitro microphysiological model of the human skin.
Detection of driver pathways using mutated gene network in cancer
Authors: Li Feng , Gao Lin , Ma Xiaoke , Yang Xiaofei
Published: April 2016

A mutated gene network is constructed based on a new mutual exclusivity index and coverage for detecting driver pathways.
Single layer nano graphene platelets derived from graphite nanofibres
Authors: Huang Kai , Delport Géraud , Orcin-Chaix Lucile , Drummond Carlos , Lauret Jean-Sebastien , Penicaud Alain
Published: April 2016

Fully exfoliated single layer nanographenes are obtained by reductive dissolution of graphite nanofibre intercalation compounds. Strong photoluminescence is observed upon air exposure of nanographenide solutions.
An efficient lactamisation/N-acyliminium Pictet–Spengler domino strategy for the diasteroselective synthesis of polyhydroxylated quinoxalinone, β-carboline and quinazolinone derivatives
Authors: Subba Reddy B. V. , Reddy B. P. , Reddy P. V. G. , Siriwardena A.
Published: April 2016

A novel Pictet–Spengler cascade sequence has been developed. It involves a lactamisation/N-acyliminium key step and gives a diverse set of sugar-derived fused polycyclic heterocycles diastereoselectively and efficiently.
Divergent copper-mediated dimerization and hydroxylation of benzamides involving C–H bond functionalization
Authors: Wang Mingliang , Hu Yimin , Jiang Zhe , Shen Hong C. , Sun Xun
Published: April 2016

Copper-mediated C–H activation of aminoquinoline benzamides to give either hydroxylation or dimerization compounds were investigated.
Gold nanoparticle-conjugated heterogeneous polymer brush-wrapped cellulose nanocrystals prepared by combining different controllable polymerization techniques for theranostic applications
Authors: Hu Hao , Hou Xiu-Ju , Wang Xiao-Chen , Nie Jing-Jun , Cai Qing , Xu Fu-Jian
Published: April 2016

Spindly cellulose nanocrystals were coated with Au nanoparticle-conjugated heterogeneous polymer brushes prepared via different controllable polymerization.
Characterization of hetero-block copolymers by the log-normal distribution model
Authors: Monteiro Michael J. , Gavrilov Mikhail
Published: April 2016

Fitting multiple and of different chemical composition molecular weight distributions using the log-normal distribution (LND) model.
O-Nitrobenzyl-alt-(phenylethynyl)benzene copolymer-based nanoaggregates with highly efficient two-photon-triggered degradable properties via a FRET process
Authors: Zhao Hui , Hou Bing , Tang Yufu , Hu Wenbo , Yin Chao , Ji Yu , Lu Xiaomei , Fan Quli , Huang Wei
Published: April 2016

This paper reports on O-nitrobenzyl-alt-(phenylethynyl)benzene copolymer-based nanoaggregates which could be efficiently degraded under two-photon excitation.
The spin-dependent transport of transition metal encapsulated B40fullerene
Authors: Wang Wei , Guo Yan-Dong , Yan Xiao-Hong
Published: April 2016

Two-probe systems of transition metal atom (X)-encapsulated B40fullerene contacted with Au electrodes, where X = Fe, Mn, Ni, and Co.
Low-k and superior comprehensive property hybrid materials of a fluorinated polyimide and pure silica zeolite
Authors: Huang Zhenxun , Zhao Jianqing
Published: April 2016

FPI/A-PSZN hybrid materials with low-k and superior comprehensive properties are potentially useful in the microelectronic industry.
Influence of original and simulated microscopic units on SHG response in semiorganic NLO materials
Authors: Hou Dianwei , Lei Bing-Hua , Pan Shilie , Zhang Bingbing , Yang Zhihua
Published: April 2016

The SHG effect increase was explored by combining the electronic structure, SHG density, molecular orbital analysis and hyperpolarizability density.
Transition of resistive switching behavior for a HfOx film induced by Ni doping
Authors: Guo Tingting , Tan Tingting , Liu Zhengtang , Wu Zhihui
Published: April 2016

In this work, Ni-doped HfOx films with different Ni doping concentrations were fabricated and the chemical bonding states as well as the resisistive switching characteristics were investigated.
Cetyltrimethylammonium bromide (CTAB) promote amyloid fibril formation in carbohydrate binding protein (concanavalin A) at physiological pH
Authors: Khan Javed Masood , Khan Mohd Shahnawaz , Ali Mohd Sajid , Al-Shabib Nasser Abdulatif , Khan Rizwan Hasan
Published: April 2016

Low concentration of CTAB provoked cross β-sheet formation whereas high concentrations of CTAB direct to alpha helix induction in Con A.
Discovering lipid phenotypic changes of sepsis-induced lung injury using high-throughput lipidomic analysis
Authors: Liang Qun , Liu Han , Jiang Yan , Xing Haitao , Zhang Tianyu , Zhang Ai-Hua
Published: April 2016

The aim of this study was to use lipidomics to identify lipid molecules that could predict patients with sepsis-induced lung injury.
Multi-pathlength method to improve the spectrometric analysis accuracy based on “M + N” theory
Authors: Li Gang , Luo YongShun , Li Zhe , Li ZeYun , Lin Ling
Published: April 2016

In order to verify the effectiveness of three application methods, an experiment was done to predict the concentration of intra-lipids by single pathlength and multi-pathlength modelling.
Efficient alternating copolymerization of sulfur dioxide with cyclohexene oxide and a mechanistic understanding
Authors: Zhi Yunfei , Wu Jing , Ma Aihua , Tian Na , Shan Shaoyun , Jia Qingming , Su Hongying
Published: April 2016

This work describes an efficient alternating copolymerization of sulfur dioxide with cyclohexene oxide using salenCrIIICl as catalyst. The effect of the temperature, time, and type of catalyst on the overall activity and selectivity was investigated.
Improving the mechanical stability of proton conducting SPEEK membranes by in situ precipitation of zirconium phosphate phenylphosphonates
Authors: Pica Monica , D'Amato Roberto , Donnadio Anna , Capitani Donatella , Troni Elisabetta , Zuccaccia Cristiano , Casciola Mario
Published: April 2016

Zirconium phosphate phenylphosphonate nanoparticles improve mechanical properties and the stability of the proton conductivity of composite SPEEK membranes.
Growth and photoluminescence of oriented MoSe2nanosheets produced by hot filament CVD
Authors: Wang B. B. , Ostrikov K. , van der Laan T. , Zheng K. , Shao R. , Zhu M. K. , Zou S. S.
Published: April 2016

Oriented MoSe2nanosheets with varying layers and structures were synthesized on silicon substrates by hot filament chemical vapour deposition in a nitrogen environment using MoO3and Se powders as precursors.
A quadrupolar two-photon fluorescent probe for in vivo imaging of amyloid-β plaques
Authors: Heo Cheol Ho , Sarkar Avik Ranjan , Baik Sung Hoon , Jung Tae Sung , Kim Jeong Jin , Kang Hyuk , Mook-Jung Inhee , Kim Hwan Myung
Published: April 2016

A quadrupolar two-photon fluorescent probe for in vivo imaging of amyloid-β plaques is reported.
Dynamically controlled deposition of colloidal nanoparticle suspension in evaporating drops using laser radiation
Authors: Ta V. D. , Carter R. M. , Esenturk E. , Connaughton C. , Wasley T. J. , Li J. , Kay R. W. , Stringer J. , Smith P. J. , Shephard J. D.
Published: April 2016

Particle deposition in a homogeneous or arbitrary distribution can be obtained by direct exposure to an infrared laser beam.
Edge-selectively antimony-doped graphene nanoplatelets as an outstanding counter electrode with an unusual electrochemical stability for dye-sensitized solar cells employing cobalt electrolytes
Authors: Kim Hong Mo , Jeon In-Yup , Choi In Taek , Kang Sung Ho , Shin Sun-Hee , Jeong Hu Young , Ju Myung Jong , Baek Jong-Beom , Kim Hwan Kyu
Published: April 2016

Edge-selectively antimony-doped graphene nanoplatelet electrocatalysts exhibited an extremely low charge transfer resistance with “zero-loss stability” for a cobalt redox couple.
Nanohybrid polymer brushes on silica for bioseparation
Authors: Jiang Lingdong , Bagán Héctor , Kamra Tripta , Zhou Tongchang , Ye Lei
Published: April 2016

Surface-initiated atom transfer radical polymerization and click chemistry are used to synthesize temperature-responsive polymer brushes for glycoprotein separation.
High-performance bipolar host materials for blue TADF devices with excellent external quantum efficiencies
Authors: Kang Ju Sik , Hong Tae Ryang , Kim Hyung Jong , Son Young Hoon , Lampande Raju , Kang Byoung Yeop , Lee Chiho , Bin Jong-Kwan , Lee Bang Sook , Yang Joong Hwan , Kim JinWuk , Park Sungnam , Cho Min Ju , Kwon Jang Hyuk , Choi Dong Hoon
Published: April 2016

2CzPN-based blue TADF-OLED devices with a ZDN host showed a remarkably high external quantum efficiency of 25.7%.
Collagen Microspheres: A Three-Dimensional Culture System for Notochordal Cells
Authors: Yuan Minting , Yeung Chiu Wai , Au Tiffany , Chan Danny , Cheah Kathryn S. E. , Chan Barbara P.
Published: April 2016

Introduction

The notochord plays a crucial role in the formation and patterning of axial skeleton. Notochordal cells (NCCs) have drawn increasing attentions from developmental biology, stem cells research and tissue engineering in these years as they are important in development and maintenance of NP. However, it is difficult to maintain their survival and phenotype in 2D culture for long term, making it difficult to understand their fate and functional maintenance during intervertebral disc development. This project is to develop a better culture system that is able to maintain the survival and phenotypes of NCCs.

Materials and Methods

Foxa2mNE–Cre/Z/EG heterozygous embryos were developed and NCCs could be identified EGFP signal. NCC isolated with or without FACS-sorting, notochord segments at the anterior, trunk and posterior regions, and whole notochord pulled out from embryo were microencapsulated respectively in type I collagen microspheres and cultured for up to 1 month before characterization for survival and phenotype maintenance.

Results

EGFP signal of sorted cells could be maintained after 4 weeks. The EGFP positive cells in collagen microspheres showed colocalization with foxa2 and brachyury, which were considered as markers of NCCs, indicated that they were still NCCs. However, areas with these merged were very rare. EGFP signal of unsorted cells could be maintained and clusters of EGFP positive cells were showed during 4-weeks culture. When the notochord segments were cultured in collagen microspheres, clusters of round EGFP positive cells co-expressing with major NCC markers were found after 1 month, suggesting that NCC phenotype was maintained. Moreover, the number of EGFP positive cells was increased during culture. In the other hand, when the notochord was cultured in collagen microsphere, it started pinching and the pinching region seemed growing at day 28.

Conclusion

Type I collagen microspheres present a potential culture system for increasing cell number with EGFP positive signal for at least 4 weeks. NCCs entrapped in type I collagen microspheres could maintained the NCCs phenotype. Comparing with FACS-sorted NCCs, there was increasing number of NCCs in unsorted group, segment culture and whole notochord culture, suggesting that the presence of niche cells, extracellular matrix, and native configuration of notochord may be important to support NCCs growth and phenotype maintenance.

Acknowledgments

This work was supported by AoE (AoE/M-04/04), TBRS (T12–708/12-N) and AOSPN (SRN_2011_14). Disclosures: None
The Species-Specific Regenerative Effect of Notochordal Cell-conditioned Medium on Degenerated Human Nucleus Pulposus Cells
Authors: Bach Frances , de Vries Stefan , van Heel Ferdi , Krouwels Anita , Creemers Laura , Ito Keita , Meij Björn , Tryfonidou Marianna
Published: April 2016

Introduction

During human intervertebral disc (IVD) degeneration, the main cell type shifts from notochordal cells (NCs) to nucleus pulposus cells (NPCs). NCs secrete factors with regenerative potential, making them an interesting focus for regenerative strategies. Research into these strategies employs non-human donors due to easy availability of their NC-rich nucleus pulposus (NP) tissue. To determine the feasibility of translating these strategies toward clinical application, this study aimed to delineate whether NC-secreted factors of different species have a cross-species regenerative effect on degenerated human NPCs.

Material and Methods

Healthy human, porcine and canine NC-rich NP tissues (derived from Thompson grade I IVDs) were compared by histology, collagen, DNA and glycosaminoglycan (GAG) content per mg wet tissue weight. NP tissue of each species was cultured for 4 days and NC-conditioned medium (NCCM) was collected. Human NPC micro-aggregates from degenerated IVDs (Thompson grade III) were cultured for 28 days in human, canine or porcine NCCM. GAG content (corrected for DNA) was determined, and Safranin O/Fast Green staining, collagen type I, II and X IHC and gene expression profiling were performed.

Results

Canine and porcine NPs were richer in NCs than human NPs. Human NPs contained the highest collagen content, whereas the DNA and GAG content of canine NPs was significantly higher than human or porcine NPs. NCCM from all species significantly increased the DNA and GAG content of the human NPC micro-aggregates. Porcine and canine NCCM were significantly more potent than human NCCM in inducing GAG deposition, whereas only human NCCM induced collagen type II production.

Conclusion

Secreted factors from human, canine and porcine NC-rich NPs exerted regenerative effects on human NPCs, indicating a cross-species effect. The species-specific NP properties appear to influence the regenerative capacity of NCCM on human NPCs. Bioactive compound(s) with treatment potential for human IVD degeneration are present in NCCM of different species, implying that strategies based on NC-technology employing canine or porcine models have potential for successful translation into humans.

Acknowledgments

This work was supported by an AOSpine Research Network grant (SRN2011_11) and the Dutch Arthritis Foundation (LLP22).
TGFbeta Cross-linked to Microcarriers as Injectable IVD Therapy in Dogs and Humans
Authors: Bertolo Alessandro , Steffen Frank , Arcolino Fanny , Capossela Simona , Taddei Anna Rita , Baur Martin , Poetzel Tobias , Stoyanov Jivko
Published: April 2016

Introduction

Studies designed to use large canine models and translate the results to human practice must take into account the ex vivo and in vitro differences in the bone marrow derived stem cells(MSCs) and their response to a covalently bound growth factor.

Methods

We analyze the activity of transforming growth factor-β1 (TGF-β1) covalently immobilized on microcarriers either by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS) or riboflavin/UV (RB/UV) light-mediated cross-linking. Collagen microcarriers cross-linked with TGF-β1 were used for chondrogenic differentiation of human or canine mesenchymal stem cells. Evaluation methods included cell viability test, chondrogenic marker expression (aggrecan and collagen type I and type II), histological detection of proteoglycans, and immunohistochemical analysis.

Results

Cross-linking strengthened the collagen structure of the microcarriers and reduced collagenase-mediated degradation. Chondrogenic differentiation of MSCs was induced by TGF-β1 cross-linked on microcarriers, promoting gene expression and protein accumulation of aggrecan and collagen type I and type II, as well as proteoglycans. Cross-linking by RB/UV enhanced chondrogenesis more than any other group. In addition, cross-linking reduced scaffold shrinkage exerted by MSCs during chondrogenesis, a desirable feature for microcarriers if used as tissue defect filler.

Conclusion

Cross-linking of TGF-β1 to collagen microcarriers supported chondrogenesis. Current veterinary clinical trial will show if such approach will lead a step closer to development of a cost-effective and locally acting device for cell-based therapy.
Compression Loading Induced Cellular Stress Response of Intervertebral Disc Cells in Organ Culture
Authors: Chooi Wai Hon , Chan Samantha C. W. , Gantenbein Benjamin , Chan Barbara P.
Published: April 2016

Introduction

Mechanical stress is often associated to interverterbal disc (IVD) degeneration and the effect of mechanical loading on IVD has been studied and reviewed.1,2 Previously, expression of heat shock proteins, HSP70 and HSP27 has been found in pathological discs.3 However, there is no direct evidence on whether IVD cells respond to the mechanical loading by expression of HSPs. The objective of this study is to investigate the stress response of IVD cells during compressive loading in an organ culture.

Materials and Methods

Fresh adult bovine caudal discs were cultured with compressive loading applied at physiological range. Effect of loading type (static and dynamic) and repeated loading (2 hours per day for 2 days) were studied. Nucleus pulposus (NP) and annulus fibrosus (AF) of the IVD were retrieved at different time points: right after loading and right after resting. Positive control discs were heat shocked (43°C). Cell activity was assessed and expression of stress response genes (HSP70 and HSF1) and matrix remodeling genes (ACAN, COL2, COL1, ADAMTS4, MMP3 and MMP13) were studied.

Results

Cell activity was maintained in all groups. Both NP and AF expressed high level of HSP70 in heat shock groups, confirming their expression in response to stress. In NP, expression of HSP70 was up-regulated after static loading and dynamic loading with higher fold change was observed after static loading. During repeated loading, HSP70 appeared to be upregulated right after loading and decreased after resting. Such trend was not observed in AF and HSF1 levels. Expressions of matrix remodeling genes did not change significantly with loading except ADAMTS4 decreased in AF during static loading.

Conclusion

This study demonstrated that NP cells upregulate expression of HSP70 in response to loading induced stress without changing cell activity and matrix remodeling significantly.

Acknowledgments

This project was funded by AO Spine (AOSPN) (grant number: SRN_2011_14) and a fellowship exchange award by AO Spine Scientific Research Network (SRN). References Chan SC, Ferguson SJ, Gantenbein-Ritter B. The effects of dynamic loading on the intervertebral disc. Eur Spine J 2011;20(11):1796–1812 Neidlinger-Wilke C, Galbusera F, Pratsinis H, et al. Mechanical loading of the intervertebral disc: from the macroscopic to the cellular level. Eur Spine J 2014;23(Suppl 3):S333–S343 Sharp CA, Roberts S, Evans H, Brown SJ. Disc cell clusters in pathological human intervertebral discs are associated with increased stress protein immunostaining. Eur Spine J 2009;18(11):1587–1594
Aberrant DNA Methylation Important Factor in Intervertebral Disc Pathophysiology
Authors: Chutkan Norman , Fulzele Sadanand
Published: April 2016

Introduction

Intervertebral disc (IVD) degeneration is one of the most important aging health problems and a leading cause of disability. However, little is known about pathophysiology of this condition. This study investigates the role of epigenetic factor in intervertebral disc gene regulation.

Material and Methods

IVD tissues were obtained from donors undergoing anterior lumbar discectomy procedures. The collected tissues were minced and immediately used for cell isolation by sequential enzyme digestion. The demethylation agent 5-azacytidine (5-AzaC) was used to inhibit DNA methylation in IVD cells. The cells were then analyzed for change in DNA methylation using Methyl-Profiler PCR array. We used Methyl-Profiler PCR array containing developmental and cell cycle regulatory genes. The Methyl-Profiler PCR arrays were performed as per manufacturer's instructions. Briefly, the cells were treated with and without 5-azaC for 4 days, and genomic DNA were isolated. Then the equal amounts of genomic DNA were overnight digested with methyl-specific restriction digestion enzyme, nonspecific restriction enzyme, a combination of methyl specific and non-specific, and a control mock digestion. Following digestion, real time PCR was performed using biorad-MyiQ cycler. The data was analyzed according to manufacturer's instructions.

Results

Methyl-Profiler PCR array showed novel genes hypo-methylated following 5-azacytidine treatment. We found that around 6 genes' promoters differentially methylated after experimental demethylation in IVD cells. The genes differentially methylated are CCND2, CDH1, HIC1, MGMT, PRDM2 and TP73. Real time PCR showed these genes were differentially regulated relative to control.

Conclusion

There are reports that these differentially methylated genes have a role in various age related degenerative diseases. We speculate that these genes may also play an important role in intervertebral disc degeneration. It would be interesting to investigate the expression of these genes and their methylation pattern in aging human degenerative IVD. In summary, our results suggest that demethylation of IVD cells may be one of the factors involved in IVD degeneration. Further studies are required to confirm our findings and to better understand the effects of aberrant DNA methylation on IVD biology and disease pathophysiology.
Mesenchymal Stem Cells Harvested from Vertebral Body and Iliac Crest Bone Marrow: Are They Equally Good? Comparison of Two Tissues and Two Harvesting Techniques
Authors: Fragkakis Evangelos M. , Jones Elena , Pountos Ippokratis , Giannoudis Peter
Published: April 2016

Introduction/Objectives

Clinical applications of Mesenchymal Stem Cells (MSCs) harvested from bone marrow (BM) are well established. Iliac crest (IC) is considered to be the gold standard. Vertebral body (VB) BM represents an alternative source, promoting bone fusion in spinal operations. Existing literature, comparing ICBM and VBBM MSCs, presents limitations. Density gradient centrifugation (lymphoprep, Lmp) for BM processing is used, despite the known effectiveness on ICBM, of the red cell lysis technique (ammonium chloride, AC). This study compared the yield and differentiation capacities of MSCs in VBBM and ICBM, and evaluated the effectiveness of two harvesting procedures on both BM sources.

Patient/Methods

Eighteen patients undergoing idiopathic scoliosis surgery were recruited (median age 15.4 years, range 13–17). BM was aspirated intraoperatively from IC (10ml) and 12th thoracic vertebra (10ml). Aspirates were processed using AC and Lmp. MSCs were enumerated using standard CFU-F assay. Colony areas, MSCs doubling rates and outcomes of the differentiation assays were statistically analyzed.

Results

AC yielded more leukocytes from ICBM (23x106cells/ml), compared with VBBM (14.4x106cells/ml, p = 0.013). Lmp yielded similar numbers of mononuclear cells from ICBM (7.5x106cells/ml) and VBBM (5x106cells/ml). More MSCs were harvested from 1ml of BM aspirate using AC technique, from both anatomical sites (IC: Lmp 199MSCs/ml, AC 367MSCs/ml, p = 0.012; VB: Lmp 296MSCs/ml, AC 440MSCs/ml, p = 0.043). There were no significant differences in MSC numbers/ml and average colony areas between the sites (IC: AC 14mm2, Lmp 15mm2; VB: AC 11mm2, Lmp 13mm2) or MSCs doubling rates in days (IC: AC 2, Lmp 1.9, VB: AC 1.9, Lmp 1.8). Calcium production, after 14 days of osteogenic induction, was higher in VBBM MSCs with both harvesting methods (AC: 15-fold, p = 0.003, Lmp: 6.6-fold). Adipogenic differentiation, measured by Nile-Red absorbance on day 21 post-induction, was not significantly different between two BM sources. Glycosaminoglycan (GAG) production, after 21 days of chondrogenic induction, was higher in VBBM MSCs (AC 2.1μg/ml, Lmp 3.7μg/ml) compared with ICBM (AC 1.2μg/ml, Lmp 2.9μg/ml), but not statistically significant.

Conclusion

AC results in more effective MSC isolation and does not affect MSCs’ proliferation and differentiation. Compared with ICBM, VBBM is richer in MSCs with osteo- and chondrogenic differentiation capacities and can therefore represent the first choice for MSC harvesting in spinal surgery.
Improvement of Bovine Nucleus Pulposus Cells Isolation Leads to Identification of Three Phenotypically Distinct Cell Subpopulations
Authors: Molinos Maria , Almeida Catarina , Gonçalves Raquel , Barbosa Mário
Published: April 2016

Introduction

Strategies to promote intervertebral disc (IVD) regeneration have been hindered by the lack of knowledge of IVD fundamental cellular/molecular components. One of the key points to be addressed is the characterization of nucleus pulposus (NP) cell population(s). This study establishes an improved method for bovine NP (bNP) cell isolation, whose procedure is still not consensual among the literature, allowing a thorough characterization of cell (sub)populations that exist in the young NP.

Material and Methods

bNP was digested with distinct enzymes (collagenase-type-I, collagenase-type-II, and collagenase- type-XI) at different concentrations (0.5, 1.0, and 2.0 mg/mL), for 4 and 19 hour. Cell yield, viability/apoptosis, and morphology were analyzed by flow cytometry and imaging flow cytometry. Identification of cell subpopulations within NP and its phenotype was investigated by assessing expression of CD29, CD44, CD45, CD34, CD146, and Brachyury.

Results

It was found that bNP cells present a similar morphology independently of the digestive enzyme used. However, cell yield was greatly improved by Coll-XI (2 mg/mL) treatment for a short digestion period. Interestingly, three subpopulations, with different sizes and auto-fluorescence, were consistently identified by flow cytometry. And crucially, differential expression of cell markers was found among these subpopulations.

Conclusion

This study demonstrated that collagenase-type-XI is an efficient enzyme that is used for digesting bNP. And most importantly, three phenotypically distinct subpopulations of cells where identified within the bNP. Such knowledge is key for a better understanding of NP cell biology and its potential endogenous regenerative capacity.
Modic Changes in Lumbar Spine: Prevalence and Genetic Association Analysis through Single Neucleotide Poymorphism Analysis of 71 Polymorphisms
Authors: Rajasekaran S. , Shetty Ajoy Prasad , Kanna Rishi
Published: April 2016

Introduction

A significant association between non-specific low back pain and Modic changes (MC) have been described. We hypothesied that a single nucleotide polymorphisms(SNP) may be involved in matrix and bone metabolism associated changes with modic changes. The present study purports to evaluate the prevalence of MC and the role of genetic polymorphisms in 40 candidate genes (including 71 SNPs).

Methods

The study was a cross sectional study performed on 809 patients irrespective of back pain status. Assessment was performed with MRI evaluation and genetic association analysis of 71 SNPs belonging to 40 genes. Inclusion criteria for patients were both sexes, age of 18 to 70 years, no evidence of other spinal diseases, previous surgery & trauma. The MRI included T1 and T2 sagittal sections of the lumbar spine. The presence and type of Modic changes & the genetic association analysis of 71 SNPs was made with reference to the presence or absence of Modic Changes. Genotyping of SNPs was performed using the Sequenome® platform.

Results

809 individuals of mean age 36.7 ± 10.8 years were studied. Based on the presence of MC, the group was divided into 702 controls and 107 cases. 64 patients had single level MC (7.9%), 32 had double level (3.9%), 5 had three and four level MC (0.6%) and one had multi-level MC. Modic changes were identified in 251 of the 1070 endplates (EP). MC was more commonly situated in the lower EP (149, 59.4%) than in the upper EP (102, 40.6%). L4–5 EP were the most commonly affected level ( n = 77, 30.7%) followed by L5-S1 (26.3%), L3–4 (23.9%), L2–3 (12.4%) and L1–2 (6.8%). Type 2 MC was the most commonly observed pattern ( n = 206, 82%), followed by Type 1 ( n = 27, 10.8%) and Type 3 ( n = 18, 7.2%). Mirroring endplate MC was observed in 75 discs (29.8%) among the total 535 discs. SNPs of Vitamin D receptor gene (VDR) and Matrix Metallo proteinase (MMP20) ( p = 0.03) were significantly associated with MC. SNP of cyclo-oxygenase gene(COX2) and Insulin Growth Factor Receptor(IGF1R) ( p = 0.03) were significantly associated with Type 2 MC.

Conclusion

The study identifies genetic polymorphisms of VDR, MMP 20, COX 2 and IGF1R to be significantly associated with MC and have not been reported previously. Understanding the etiopathogenetic of Modic changes would help us to plan preventive and therapeutic strategies
Phenotype Standardization is the Urgent Need in Genetic Association Studies of Disc Degeneration - Results of Analysis of Genetic Association of 71 SNPs with Highly Specific Phenotypes in 809 Subjects
Authors: Rajasekaran S. , Kanna Rishi M. , Natesan Senthil , Muthurajan Raveendran , Ranjani Veera , Cheung Kenneth M. C. , Chan Danny , Kao Patrick Y. P. , Yee Anita , Prasad Ajoy
Published: April 2016

Introduction

Although the influence of genetics on etio-pathogenesis of disc degeneration is well recognized, there is a wide variation in the different genes observed to have association with disc degeneration. The clinical and radiographic features of disc degeneration used as phenotypes are quite variable in previously published studies. It is unclear whether the variations in genetic association studies depict variations in ethnicity or selection of phenotypes.

Methods

The purpose of the study was to evaluate the allelic diversity of 71 SNPs related to six different MRI markers of lumbar degenerative disc disease (annular tear, Pfirmann's grading, Schmorl's nodes, Modic changes, Total Endplate Damage score and disc bulge) in a single study population, and analyze how genetic associations can vary in the same study subjects with the choice of phenotype, based on age and sequence of selection of study subjects. Genotyping of cases and controls was performed on Genome wide SNP array to identify potentially associated disease loci. The results from the GWAS were then used to facilitate SNP selection and genotype validation was conducted using Sequenom based genotyping.

Results

The mean age of 809 subjects (M: 455, F:354) was 36.76 ± 10.8 years (range 10 – 80). Highly significant association ( p < 0.01) was observed with three SNPs of CILP for disc bulge and rs2249350 of ADAMTS5 and rs11247361IGF1R for annular tears. Significant association ( p < 0.05) was observed with polymorphisms of VDR and MMP20 for Modic changes, three SNPs of MMP20 for Schmorl's node and SNPs of CALM1 and FN1 for Pfirmann's grading. None of the SNPs had significant association with TEPS. Subgroup analysis based on age (<30, 30–40 and > 40 years) showed new set of genetic associations for all the six radiographic parameters. Similarly the population was divided into two groups based on numerical order and the association patterns completely differed as compared with the total group.

Conclusions

In the same study population with DDD, SNP associations completely change when phenotypes changed. Variations in age, sequence of study subjects, number of population apart from radiographic description of DDD significantly change the genetic association. Based on current results, it is difficult to consider one set of genes as responsible for disc degeneration considering these variations. Our study results demand an urgent need for standardizing the description of DDD, phenotype selection, and study criteria for genetic association studies.
Discectomy-Related Information on the Internet: Does the Quality Follow the Surge?
Authors: Sheridan Gerard , Elhassan Yahya , Mujtaba Nassiri , Mugtaba Sulman , Kiely Pat , Noel Jacques
Published: April 2016

Introduction

Over 2.5 billion users search the internet world wide. 70%- 80% look at health related information, yet no standards exist regarding the publication of medical literature on the internet. The quality of health information is of much concern and an emphasis on appraisal of internet Web sites is needed. We aim to look at the quality of health information on the internet relating to discectomy using various standardised tools and scoring systems.

Material and Methods

The three most commonly used search engines were identified and a search for “Discectomy” was performed on each. Two reviewers categorized the most popular Web sites according to their types and the quality of each was assessed using recognized scoring systems including the Journal of the American Medical Association (JAMA) benchmark, DISCERN and a novel Discectomy-specific content score. The quality of the information was also assessed according to the presence or absence of the Health on the Net (HON) code, a reported quality-assurance marker.

Results

Results confirmed that 53 websites were identified, and analyzed. Commercial Web sites were predominant at 24, 7 were governmental, 6 were produced by physicians, none were produced by allied health professionals, 3 were academic, 4 were public health information Web sites, 4 were attached to social media and discussion groups, 3 were media related, and 2 were unspecified. Using the 3 scoring systems mentioned above as a quality marker, internet sites with a 'Health on the Net’ (HON) code demonstrated significantly higher quality than those without the code ( p < 0.0001). Public health information Web sites attained the highest overall DISCERN and Discectomy-specific scores followed by the Governmental & Non-Profit Organizations Web sites.

Conclusion

The overall quality of information regarding discectomy remains poor and variable despite an exponential increase in the number of users and Websites, with a slight trend toward improvement. Only 20% to 30% are of good quality, compared with that 10 years ago (<10%). Presence of Health on the Net code is a very reliable marker for health information quality.