bluuebunny/crossref_metadata_2025 huggingface dataset:
Structure and function of chicken interleukin-1 beta mutants: uncoupling of receptor binding and in vivo biological activity
Authors: Chen Wen-Ting , Huang Wen-Yang , Chen Ting , Salawu Emmanuel Oluwatobi , Wang Dongli , Lee Yi-Zong , Chang Yuan-Yu , Yang Lee-Wei , Sue Shih-Che , Wang Xinquan , Yin Hsien-Sheng
Published: June 2016

Receptor-binding and subsequent signal-activation of interleukin-1 beta (IL-1β) are essential to immune and proinflammatory responses. We mutated 12 residues to identify sites important for biological activity and/or receptor binding. Four of these mutants with mutations in loop 9 (T117A, E118K, E118A, E118R) displayed significantly reduced biological activity. Neither T117A nor E118K mutants substantially affected receptor binding, whereas both mutants lack the IL-1β signaling in vitro but can antagonize wild-type (WT) IL-1β. Crystal structures of T117A, E118A and E118K revealed that the secondary structure or surface charge of loop 9 is dramatically altered compared with that of wild-type chicken IL-1β. Molecular dynamics simulations of IL-1β bound to its receptor (IL-1RI) and receptor accessory protein (IL-1RAcP) revealed that loop 9 lies in a pocket that is formed at the IL-1RI/IL-1RAcP interface. This pocket is also observed in the human ternary structure. The conformations of above mutants in loop 9 may disrupt structural packing and therefore the stability in a chicken IL-1β/IL-1RI/IL-1RAcP signaling complex. We identify the hot spots in IL-1β that are essential to immune responses and elucidate a mechanism by which IL-1β activity can be inhibited. These findings should aid in the development of new therapeutics that neutralize IL-1 activity.
Carbon nanotubes affect the toxicity of CuO nanoparticles to denitrification in marine sediments by altering cellular internalization of nanoparticle
Authors: Zheng Xiong , Su Yinglong , Chen Yinguang , Wan Rui , Li Mu , Huang Haining , Li Xu
Published: June 2016

Denitrification is an important pathway for nitrate transformation in marine sediments and this process has been observed to be negatively affected by engineered nanomaterials. However, previous studies only focused on the potential effect of a certain type of nanomaterial on microbial denitrification. Here we show that the toxicity of CuO nanoparticles (NPs) to denitrification in marine sediments is highly affected by the presence of carbon nanotubes (CNTs). It was found that the removal efficiency of total NOX−-N (NO3−-N and NO2−-N) in the presence of CuO NPs was only 62.3%, but it increased to 81.1% when CNTs appeared in this circumstance. Our data revealed that CuO NPs were more easily attached to CNTs rather than cell surface because of the lower energy barrier (3.5 versus 36.2 kT). Further studies confirmed that the presence of CNTs caused the formation of large, incompact, non-uniform dispersed and more negatively charged CuO-CNTs heteroaggregates and thus reduced the nanoparticle internalization by cells, leading to less toxicity to metabolism of carbon source, generation of reduction equivalent and activities of nitrate reductase and nitrite reductase. These results indicate that assessing nanomaterial-induced risks in real circumstances needs to consider the “mixed” effects of nanomaterials.
Ultrasound characterization of the mastoid for detecting middle ear effusion: A preliminary clinical validation
Authors: Chen Chin-Kuo , Fang Jui , Wan Yung-Liang , Tsui Po-Hsiang
Published: June 2016

Ultrasound detection of middle ear effusion (MEE) is an emerging technique in otolaryngology. This study proposed using ultrasound characterization of the mastoid to noninvasively measure MEE-induced mastoid effusion (ME) as a new strategy for determining the presence of MEE. In total, 53 patients were enrolled (Group I: normal, n = 20; Group II: proven MEE through both otoscopy and tympanometry, n = 15; Group III: patients with MEE having effusions observed during grommet surgery, n = 18). A 2.25-MHz delay-line transducer was used to measure backscattered signals from the mastoid. The Nakagami parameter was estimated using the acquired signals to model the echo amplitude distribution for quantifying changes in the acoustic structures of mastoid air cells. The median Nakagami parameter and interquartile range were 0.35 (0.34–0.37) for Group I, 0.39 (0.37–0.41) for Group II and 0.43 (0.39–0.51) for Group III. The echo amplitude distribution observed for patients with MEE was closer to Rayleigh distribution than that without MEE. Receiver operating characteristic (ROC) curve analysis further revealed that the area under the ROC was 0.88, sensitivity was 72.73%, specificity was 95% and accuracy was 81.13%. The proposed method has considerable potential for noninvasive and comfortable evaluation of MEE.
The structural alteration of gut microbiota in low-birth-weight mice undergoing accelerated postnatal growth
Authors: Wang Jingjing , Tang Huang , Wang Xiaoxin , Zhang Xu , Zhang Chenhong , Zhang Menghui , Zhao Yufeng , Zhao Liping , Shen Jian
Published: June 2016

The transient disruption of gut microbiota in infancy by antibiotics causes adult adiposity in mice. Accelerated postnatal growth (A) leads to a higher risk of adult metabolic syndrome in low birth-weight (LB) humans than in normal birth-weight (NB) individuals, but the underlying mechanism remains unclear. Here, we set up an experiment using LB + A mice, NB + A mice, and control mice with NB and normal postnatal growth. At 24 weeks of age (adulthood), while NB + A animals had a normal body fat content and glucose tolerance compared with controls, LB + A mice exhibited excessive adiposity and glucose intolerance. In infancy, more fecal bacteria implicated in obesity were increased in LB + A pups than in NB + A pups, including Desulfovibrionaceae, Enterorhabdus, and Barnesiella. One bacterium from the Lactobacillus genus, which has been implicated in prevention of adult adiposity, was enhanced only in NB + A pups. Besides, LB + A pups, but not NB + A pups, showed disrupted gut microbiota fermentation activity. After weaning, the fecal microbiota composition of LB + A mice, but not that of NB + A animals, became similar to that of controls by 24 weeks. In infancy, LB + A mice have a more dysbiotic gut microbiome compared to NB + A mice, which might increase their risk of adult metabolic syndrome.
1H-detected solid-state NMR of proteins entrapped in bioinspired silica: a new tool for biomaterials characterization
Authors: Ravera Enrico , Cerofolini Linda , Martelli Tommaso , Louka Alexandra , Fragai Marco , Luchinat Claudio
Published: June 2016

Proton-detection in solid-state NMR, enabled by high magnetic fields (>18 T) and fast magic angle spinning (>50 kHz), allows for the acquisition of traditional 1H-15N experiments on systems that are too big to be observed in solution. Among those, proteins entrapped in a bioinspired silica matrix are an attractive target that is receiving a large share of attention. We demonstrate that 1H-detected SSNMR provides a novel approach to the rapid assessment of structural integrity in proteins entrapped in bioinspired silica.
Effects of LDL Receptor Modulation on Lymphatic Function
Authors: Milasan Andreea , Dallaire François , Mayer Gaétan , Martel Catherine
Published: June 2016

Atherosclerosis is driven by the accumulation of immune cells and cholesterol in the arterial wall. Although recent studies have shown that lymphatic vessels play an important role in macrophage reverse cholesterol transport, the specific underlying mechanisms of this physiological feature remain unknown. In the current report, we sought to better characterize the lymphatic dysfunction that is associated with atherosclerosis by studying the physiological and temporal origins of this impairment. First, we assessed that athero-protected Pcsk9−/− mice exhibited improved collecting lymphatic vessel function throughout age when compared to WT mice for up to six months, while displaying enhanced expression of LDLR on lymphatic endothelial cells. Lymphatic dysfunction was present before the atherosclerotic lesion formation in a mouse model that is predisposed to develop atherosclerosis (Ldlr−/−; hApoB100+/+). This dysfunction was presumably associated with a defect in the collecting lymphatic vessels in a non-specific cholesterol- but LDLR-dependent manner. Treatment with a selective VEGFR-3 agonist rescued this impairment observed early in the onset of this arterial disease. We suggest that LDLR modulation is associated with early atherosclerosis-related lymphatic dysfunction and bring forth a pleiotropic role for PCSK9 in lymphatic function. Our study unveils new potential therapeutic targets for the prevention and treatment of atherosclerosis.
Oxidized Low-Density Lipoprotein Induces Apoptosis in Cultured Neonatal Rat Cardiomyocytes by Modulating the TLR4/NF-κB Pathway
Authors: Wang Xiantao , Sun Yuhan , Yang Huafeng , Lu Yuanxi , Li Lang
Published: June 2016

This study was designed to investigate the apoptosis induced by oxidized low-density lipoprotein (ox-LDL) in cultured neonatal rat cardiomyocytes and explore the possible mechanisms. We evaluated whether ox-LDL-induced apoptosis depended in part on the activation of toll-like receptor-4(TLR4)/Nuclear factor κB (NF-κB) signaling pathway. Cells were cultivated with and without ox-LDL. Cell apoptosis was evaluated by flow cytometry. Immunofluorescence, western blot analysis and quantitative real-time polymerase chain reaction (qRT-PCR) were conducted to assess protein or mRNA expressions. Resatorvid (TAK-242), an exogenous synthetic antagonist for TLR4, was used to inhibit TLR4 signal transduction. Dose- and time-dependent apoptotic index of cardiomyocytes occurred after ox-LDL treatment. Incubation of cardiomyocytes with ox-LDL (50 μg/mL) for 24 hours increased TLR4 and NF-κB expressions significantly. Decrease of Bcl-2/Bax protein ratio, activation of caspase-3 and 9 were also detected. Ox-LDL-induced cardiomyocyte apoptosis, TLR4 and NF-κB expressions were attenuated by pretreatment with TAK-242. In conclusion, our findings indicate that the apoptosis induced by ox-LDL in cultured neonatal rat cardiomyocytes at least in part by modulating the TLR4/NF-κB signaling pathway.
Fast, sensitive point of care electrochemical molecular system for point mutation and select agent detection
Authors: MacLeod J. A. , Nemeth A. C. , Dicke W. C. , Wang D. , Manalili Wheeler S. , Hannis J. C. , Collier G. B. , Drader J. J.
Published: June 2016

Point of care molecular diagnostics benefits from a portable battery-operated device capable of performing a fast turnaround using reliable inexpensive cartridges.
Reduction of matrix effects through a simplified QuEChERS method and using small injection volumes in a LC-MS/MS system for the determination of 28 pesticides in fruits and vegetables
Authors: Li Honge , Wang Chenchen , Zhu Qian , Du Hongxia , Guan Shuai , Wang Fengen , Zhang Wenjun , Fan Wenjing , Chen Zilei , Yang Guosheng , Aboul-Enein Hassan Y.
Published: June 2016

Reduction of matrix effects to a negligible level by using small injection volumes.
Are two better than one? Comparing intermolecular and intramolecular indicator displacement assays in pyrophosphate sensors
Authors: Liu Xuejian , Smith David G. , Jolliffe Katrina A.
Published: June 2016

Pyrophosphate recognition using indicator displacement assays is compared to that of analogous receptors with covalently attached indicators.
Wetting dynamics of a water nanodrop on graphene
Authors: Andrews Joseph Eugene , Sinha Shayandev , Chung Peter W. , Das Siddhartha
Published: June 2016

Spreading of water nanodrop on supported and unsupported graphene reveals inertia-dominated behavior.
Theoretical analysis of hot electron injection from metallic nanotubes into a semiconductor interface
Authors: Kumarasinghe Chathurangi S. , Premaratne Malin , Gunapala Sarath D. , Agrawal Govind P.
Published: June 2016

Hot electron injection from a metallic nanotube under optical illumination into a semiconductor layer in contact with its inner boundary.
Highly efficient perturbative + variational strategy based on orthogonal valence bond theory for the evaluation of magnetic coupling constants. Application to the trinuclear Cu(ii) site of multicopper oxidases
Authors: Tenti Lorenzo , Maynau Daniel , Angeli Celestino , Calzado Carmen J.
Published: June 2016

A newperturbative + variationalstrategy: a low-cost, quantitative and rational evaluation of the magnetic coupling constant in complex systems.
Chiral heterobimetallic chains from a dicyanideferrite building block including a π-conjugated TTF annulated ligand
Authors: Cui Long , Lv Zhong-Peng , Leong Chanel F. , Ru Jing , D'Alessandro Deanna M. , Song You , Zuo Jing-Lin
Published: June 2016

Two enantiopure 1D complexes were synthesized from the dicyanideferrite building block with a π-conjugated TTF annulated ligand. A field-induced magnetic phase transition is observed (TN = 4.8 K) for compound 2-(SS).
Nonlinear optical properties of intriguing Ru σ-acetylide complexes and the use of a photocrosslinked polymer as a springboard to obtain SHG active thin films
Authors: Colombo Alessia , Dragonetti Claudia , Marinotto Daniele , Righetto Stefania , Griffini Gianmarco , Turri Stefano , Akdas-Kilig Huriye , Fillaut Jean-Luc , Amar Anissa , Boucekkine Abdou , Katan Claudine
Published: June 2016

The nonlinear optics of Ru σ-acetylides have been reported.
Versatile coordination of a reactive P,N-ligand toward boron, aluminum and gallium and interconversion reactivity
Authors: Devillard M. , Alvarez Lamsfus C. , Vreeken V. , Maron L. , van der Vlugt J. I.
Published: June 2016

The unique and well-defined coordination chemistry of a reactive lutidine-derived dearomatized phosphino-amido ligand toward Group 13 complexes is presented.
Landscape geomorphic characteristic impacts on greenhouse gas fluxes in exposed stream and riparian sediments
Authors: Vidon Philippe , Serchan Satish
Published: June 2016

In forest streams, riparian zone gas emissions exceed those of in-stream locations, with most CO2eq driven by CH4 production.
Heterogeneous palladium-catalysed Catellani reaction in biomass-derived γ-valerolactone
Authors: Rasina Dace , Kahler-Quesada Arianna , Ziarelli Simone , Warratz Svenja , Cao Hui , Santoro Stefano , Ackermann Lutz , Vaccaro Luigi
Published: June 2016

Herein, we report the unprecedented use of a heterogeneous palladium catalyst for the step-economical Catellani reaction.
Differential quantification of isobaric phosphopeptides using data-independent acquisition mass spectrometry
Authors: Sidoli Simone , Fujiwara Rina , Kulej Katarzyna , Garcia Benjamin A.
Published: June 2016

Data-independent acquisition (DIA) is here applied to determine unexplored phosphorylation events on isobarically modified peptides.
Enantioselective recognition of ascorbic acid and isoascorbic acid on HS-β-cyclodextrin/gold nanoparticles/hollow carbon microspheres hybrid modified electrodes
Authors: Xu Juanjuan , Ying Quanhong , Xia Qiao , Wang Ni , Lin Xia , Fu Yingzi
Published: June 2016

The HS-β-cyclodextrin/gold nanoparticles/hollow carbon microspheres hybrids were prepared to develop a simple strategy for electrochemical chiral analysis.
DABCO-functionalized silica–copper(i) complex: a novel and recyclable heterogeneous nanocatalyst for palladium-free Sonogashira cross-coupling reactions
Authors: Hajipour Abdol Reza , Hosseini Seyed Mostafa , Mohammadsaleh Fatemeh
Published: June 2016

Nano-CuI stabilized in a functionalized-DABCO silica matrix was prepared, characterized and evaluated as a new heterogeneous nanocatalyst in copper-catalyzed Sonogashira coupling reactions.
Organocatalytic 1,3-dipolar [3+3] cycloaddition reaction of 1,2-cyclohexanedione with azides: direct access to highly substituted 1,2,3-triazoles
Authors: Xu Xianhong , Shi Zhenyan , Li Wenjun
Published: June 2016

Enamine-mediated 1,3-dipolar [3+3] cycloaddition reactions of 1,2-cyclohexanedione with azides have been developed. This strategy could generate highly substituted 1,2,3-triazoles in high yields and high levels of regioselectivities. It is demonstrated that various types of products could be obtained with different organocatalysts and substrates.
Protonation-induced red-coloured circularly polarized luminescence of [5]carbohelicene fused by benzimidazole
Authors: Sakai Hayato , Kubota Takako , Yuasa Junpei , Araki Yasuyuki , Sakanoue Tomo , Takenobu Taishi , Wada Takehiko , Kawai Tsuyoshi , Hasobe Taku
Published: June 2016

We report the first observation of red-coloured circularly polarized luminescence (CPL) of a helicene derivative (benzimidazole-fused [5]carbohelicene) induced by protonation.
Stepwise unfolding of a multi-tryptophan protein MPT63 with immunoglobulin-like fold: detection of zone-wise perturbation during guanidine hydrochloride-induced unfolding using phosphorescence spectroscopy
Authors: Mukherjee Manini , Ghosh Ranendu , Chattopadhyay Krishnananda , Ghosh Sanjib
Published: June 2016

The present article highlights the simple inexpensive and rapid technique of phosphorescence spectroscopy at 77 K that reveals the environment of all the four tryptophan residues of a protein MPT63 during guanidine hydrochloride induced unfolding.
Pulsatile release from pH triggered imidazoline switchable surfactant liposomes
Authors: Hegh Dylan Y. , Mackay Sean M. , Tan Eng Wui
Published: June 2016

The incorporation of an imidazoline (IDZ) based switchable surfactant into the lipid membrane of a liposome produces a system that can be triggered to release its solute upon pH change.
Electrospun Pd-doped mesoporous carbon nano fibres as catalysts for rechargeable Li–O2batteries
Authors: Martinez Crespiera S. , Amantia D. , Knipping E. , Aucher C. , Aubouy L. , Amici J. , Zeng J. , Francia C. , Bodoardo S.
Published: June 2016

Mesoporous carbon nanofibres doped with palladium nanoparticles (Pd CNFs) are synthesized by electrospinning with subsequent thermal treatment processes and used as electro-catalysts at the oxygen cathode of Li–O2batteries.
Nitrogen-rich hypergolic ionic salts based on (2-methyltetrazol-5-yl)diazotates
Authors: Wang Qi , Lu Huijie , Pang Fuqing , Huang Jinglun , Nie Fude , Chen Fu-Xue
Published: June 2016

Novel hypergolic energetic salts containing the (2-methyltetrazol-5-yl)diazotate anion were synthesized and exhibit attractive short ignition delay times with white fuming HNO3.
General chemistry students' conceptual understanding and language fluency: acid–base neutralization and conductometry
Authors: Nyachwaya James M.
Published: June 2016

The objective of this study was to examine college general chemistry students' conceptual understanding and language fluency in the context of the topic of acids and bases. 115 students worked in groups of 2–4 to complete an activity on conductometry, where they were given a scenario in which a titration of sodium hydroxide solution and dilute hydrochloric acid was tracked by measuring electrical conductivity. Students were asked to write a balanced equation for the reaction, provide a particulate level drawing of the reactants and products, write a net ionic equation for the reaction, predict how electrical conductivity would change with the addition of sodium hydroxide to the acid, provide a sketch of their prediction, and explain their sketch. As students worked on the activity, conversations in their groups were audio recorded. Their written responses and audio conversations were analyzed to decipher conceptual understanding and language fluency. Results showed widespread lack of conceptual understanding as well as a lack of language fluency. Students struggled with very basic ideas regarding acid–base chemistry, such as identifying the right species involved in the neutralization reaction, and providing symbolic and sub-microscopic representations (an aspect of the language of chemistry) of the acid–base reaction. Most students could not accurately predict how electrical conductivity would change as the neutralization reaction progressed. None of the groups provided an accurate sketch depicting the trend of electrical conductivity. Most of the groups did not correctly apply acid–base neutralization ideas to the context of conductometry, indicating that students were not able to transfer knowledge of acid–base neutralization to this new context.
Palladium-catalyzed regioselective and stereo-invertive ring-opening borylation of 2-arylaziridines with bis(pinacolato)diboron: experimental and computational studies
Authors: Takeda Youhei , Kuroda Akinobu , Sameera W. M. C. , Morokuma Keiji , Minakata Satoshi
Published: June 2016

A palladium catalyzed regioselective borylative ring-opening reaction of 2-arylaziridines to give β-amino-β-arylethylborates was developed.
Atomic-level organization of vicinal acid–base pairs through the chemisorption of aniline and derivatives onto mesoporous SBA15
Authors: Hamzaoui Bilel , Bendjeriou-Sedjerari Anissa , Pump Eva , Abou-Hamad Edy , Sougrat Rachid , Gurinov Andrei , Huang Kuo-Wei , Gajan David , Lesage Anne , Emsley Lyndon , Basset Jean-Marie
Published: June 2016

The design of novel heterogeneous catalysts with multiple adjacent functionalities is of high interest to heterogeneous catalysis.
Single-layered platinum nanocage: a highly selective and efficient catalyst for fuel cells
Authors: Mahata Arup , Rawat Kuber Singh , Choudhuri Indrani , Pathak Biswarup
Published: June 2016

Developing a highly efficient catalyst for the oxygen reduction reaction (ORR) is key to the performance of fuel cells for future energy applications.
The effect of subarachnoid erythrocyte lysate on brain injury: a preliminary study
Authors: Zhang Zi-Huan , Han Yan-Ling , Wang Chun-Xi , Zhou Chen-Hui , Wu Ling-Yun , Zhang Hua-Sheng , Chen Qiang , Fan Jie-Mei , Zhou Meng-Liang , Hang Chun-Hua
Published: June 2016

Abundant erythrocytes remain and lyse partially in the subarachnoid space after severe subarachnoid haemorrhage (SAH). But the effect of subarachnoid erythrocyte lysate on brain injury is still not completely clear. In this study, autologous erythrocytes (the non-lysate group) and their lysate (the lysate group) were injected separately into the cistern magna of rabbits to induce a model of experimental SAH, although the control group received isotonic sodium chloride solution instead of erythrocyte solution. Results showed that vasospasm of the basilar artery was observed at 72 h after experimental SAH, but there was no significant difference between the non-lysate group and the lysate group. Brain injury was more severe in the lysate group than in the non-lysate group. Meanwhile, the levels of peroxiredoxin 2 (Prx2), IL-6 and TNF-α in brain cortex and in CSF were significantly higher in the lysate group than those in the non-lysate group. These results demonstrated that brain injury was more likely to be caused by erythrocyte lysate than by intact erythrocytes in subarachnoid space, and inflammation response positively correlated with Prx2 expression might be involved in mechanism of brain injury after SAH.
Synthetic biology meets tissue engineering
Authors: Davies Jamie A. , Cachat Elise
Published: June 2016

Classical tissue engineering is aimed mainly at producing anatomically and physiologically realistic replacements for normal human tissues. It is done either by encouraging cellular colonization of manufactured matrices or cellular recolonization of decellularized natural extracellular matrices from donor organs, or by allowing cells to self-organize into organs as they do during fetal life. For repair of normal bodies, this will be adequate but there are reasons for making unusual, non-evolved tissues (repair of unusual bodies, interface to electromechanical prostheses, incorporating living cells into life-support machines). Synthetic biology is aimed mainly at engineering cells so that they can perform custom functions: applying synthetic biological approaches to tissue engineering may be one way of engineering custom structures. In this article, we outline the ‘embryological cycle’ of patterning, differentiation and morphogenesis and review progress that has been made in constructing synthetic biological systems to reproduce these processes in new ways. The state-of-the-art remains a long way from making truly synthetic tissues, but there are now at least foundations for future work.
Folding outer membrane proteins independently of the β-barrel assembly machinery: an assembly pathway for multimeric complexes?
Authors: Huysmans Gerard H.M.
Published: June 2016

Since the discovery of the essential role of the β-barrel assembly machinery (BAM) for the membrane insertion of outer membrane proteins (OMPs) that are unrelated in sequence, members of this universally conserved family dominate discussions on OMP assembly in bacteria, mitochondria and chloroplasts. However, several multimeric bacterial OMPs assemble independently of the catalyzing BAM-component BamA. Recent progress on this alternative pathway is reviewed here, and a model for BAM-independent assembly for multimeric OMPs is proposed in which monomer delivery to the membrane and stable prepore formation are key steps towards productive membrane insertion.
The Foundry: the DNA synthesis and construction Foundry at Imperial College
Authors: Chambers Stephen , Kitney Richard , Freemont Paul
Published: June 2016

The establishment of a DNA synthesis and construction foundry at Imperial College in London heralds a new chapter in the development of synthetic biology to meet new global challenges. The Foundry employs the latest technology to make the process of engineering biology easier, faster and scalable. The integration of advanced software, automation and analytics allows the rapid design, build and testing of engineered organisms.
SYNBIOCHEM–a SynBio foundry for the biosynthesis and sustainable production of fine and speciality chemicals
Authors: Carbonell Pablo , Currin Andrew , Dunstan Mark , Fellows Donal , Jervis Adrian , Rattray Nicholas J.W. , Robinson Christopher J. , Swainston Neil , Vinaixa Maria , Williams Alan , Yan Cunyu , Barran Perdita , Breitling Rainer , Chen George Guo-Qiang , Faulon Jean-Loup , Goble Carole , Goodacre Royston , Kell Douglas B. , Feuvre Rosalind Le , Micklefield Jason , Scrutton Nigel S. , Shapira Philip , Takano Eriko , Turner Nicholas J.
Published: June 2016

The Manchester Synthetic Biology Research Centre (SYNBIOCHEM) is a foundry for the biosynthesis and sustainable production of fine and speciality chemicals. The Centre's integrated technology platforms provide a unique capability to facilitate predictable engineering of microbial bio-factories for chemicals production. An overview of these capabilities is described.
Membrane transporters studied by EPR spectroscopy: structure determination and elucidation of functional dynamics
Authors: Mullen Anna , Hall Jenny , Diegel Janika , Hassan Isa , Fey Adam , MacMillan Fraser
Published: June 2016

During their mechanistic cycles membrane transporters often undergo extensive conformational changes, sampling a range of orientations, in order to complete their function. Such membrane transporters present somewhat of a challenge to conventional structural studies; indeed, crystallization of membrane-associated proteins sometimes require conditions that vary vastly from their native environments. Moreover, this technique currently only allows for visualization of single selected conformations during any one experiment. EPR spectroscopy is a magnetic resonance technique that offers a unique opportunity to study structural, environmental and dynamic properties of such proteins in their native membrane environments, as well as readily sampling their substrate-binding-induced dynamic conformational changes especially through complementary computational analyses. Here we present a review of recent studies that utilize a variety of EPR techniques in order to investigate both the structure and dynamics of a range of membrane transporters and associated proteins, focusing on both primary (ABC-type transporters) and secondary active transporters which were key interest areas of the late Professor Stephen Baldwin to whom this review is dedicated.
Pi sensing and signalling: from prokaryotic to eukaryotic cells
Authors: Qi Wanjun , Baldwin Stephen A. , Muench Stephen P. , Baker Alison
Published: June 2016

Phosphorus is one of the most important macronutrients and is indispensable for all organisms as a critical structural component as well as participating in intracellular signalling and energy metabolism. Sensing and signalling of phosphate (Pi) has been extensively studied and is well understood in single-cellular organisms like bacteria (Escherichia coli) and Saccharomyces cerevisiae. In comparison, the mechanism of Pi regulation in plants is less well understood despite recent advances in this area. In most soils the available Pi limits crop yield, therefore a clearer understanding of the molecular basis underlying Pi sensing and signalling is of great importance for the development of plants with improved Pi use efficiency. This mini-review compares some of the main Pi regulation pathways in prokaryotic and eukaryotic cells and identifies similarities and differences among different organisms, as well as providing some insight into future research.
The varied functions of aluminium-activated malate transporters–much more than aluminium resistance
Authors: Palmer Antony J. , Baker Alison , Muench Stephen P.
Published: June 2016

The ALMT (aluminium-activated malate transporter) family comprises a functionally diverse but structurally similar group of ion channels. They are found ubiquitously in plant species, expressed throughout different tissues, and located in either the plasma membrane or tonoplast. The first family member identified was TaALMT1, discovered in wheat root tips, which was found to be involved in aluminium resistance by means of malate exudation into the soil. However, since this discovery other family members have been shown to have many other functions such as roles in stomatal opening, general anionic homoeostasis, and in economically valuable traits such as fruit flavour. Recent evidence has also shown that ALMT proteins can act as key molecular actors in GABA (γ-aminobutyric acid) signalling, the first evidence that GABA can act as a signal transducer in plants.
Hydrogen activation by [NiFe]-hydrogenases
Authors: Carr Stephen B. , Evans Rhiannon M. , Brooke Emily J. , Wehlin Sara A.M. , Nomerotskaia Elena , Sargent Frank , Armstrong Fraser A. , Phillips Simon E.V.
Published: June 2016

Hydrogenase-1 (Hyd-1) from Escherichia coli is a membrane-bound enzyme that catalyses the reversible oxidation of molecular H2. The active site contains one Fe and one Ni atom and several conserved amino acids including an arginine (Arg509), which interacts with two conserved aspartate residues (Asp118 and Asp574) forming an outer shell canopy over the metals. There is also a highly conserved glutamate (Glu28) positioned on the opposite side of the active site to the canopy. The mechanism of hydrogen activation has been dissected by site-directed mutagenesis to identify the catalytic base responsible for splitting molecular hydrogen and possible proton transfer pathways to/from the active site. Previous reported attempts to mutate residues in the canopy were unsuccessful, leading to an assumption of a purely structural role. Recent discoveries, however, suggest a catalytic requirement, for example replacing the arginine with lysine (R509K) leaves the structure virtually unchanged, but catalytic activity falls by more than 100-fold. Variants containing amino acid substitutions at either or both, aspartates retain significant activity. We now propose a new mechanism: heterolytic H2 cleavage is via a mechanism akin to that of a frustrated Lewis pair (FLP), where H2 is polarized by simultaneous binding to the metal(s) (the acid) and a nitrogen from Arg509 (the base).
Rhomboids, signalling and cell biology
Authors: Freeman Matthew
Published: June 2016

Here, I take a somewhat personal perspective on signalling control, focusing on the rhomboid-like superfamily of proteins that my group has worked on for almost 20 years. As well as describing some of the key and recent advances, I attempt to draw out signalling themes that emerge. One important message is that the genetic and biochemical perspective on signalling has tended to underplay the importance of cell biology. There is clear evidence that signalling pathways exploit the control of intracellular trafficking, protein quality control and degradation and other cell biological phenomena, as important regulatory opportunities.
Treatment of metastatic renal cell carcinoma (mRCC) with CAIX CAR-engineered T-cells–a completed study overview
Authors: Lamers Cor H.J. , Klaver Yarne , Gratama Jan W. , Sleijfer Stefan , Debets Reno
Published: June 2016

We studied safety and proof of concept of a phase I/II trial with chimeric antigen receptor (CAR) T-cells in patients with metastatic renal cell carcinoma (mRCC). The CAR was based on the G250 mAb that recognized an epitope of carboxy-anhydrase-IX (CAIX). Twelve patients with CAIX+ mRCC were treated in three cohorts with a maximum of 10 daily infusions of 2×107 to 2×109 CAR T-cells. Circulating CAR T-cells were transiently detectable in all patients and maintained antigen-specific immune functions following their isolation post-treatment. Blood cytokine profiles mirrored CAR T-cell presence and in vivo activity. Unfortunately, patients developed anti-CAR T-cell antibodies and cellular immune responses. Moreover, CAR T-cell infusions induced liver enzyme disturbances reaching CTC grades 2–4, which necessitated cessation of treatment in four out of eight patients (cohort 1+2). Examination of liver biopsies revealed T-cell infiltration around bile ducts and CAIX expression on bile duct epithelium, adding to the notion of on-target toxicity. No such toxicities were observed in four patients that were pretreated with G250 mAb (cohort 3). The study was stopped due to the advent of competing treatments before reaching therapeutic or maximum tolerated dose in cohort 3. No clinical responses have been recorded. Despite that, from this trial numerous recommendations for future trials and their immune monitoring could be formulated, such as choice of the target antigen, format and immunogenicity of receptor and how the latter relates to peripheral T-cell persistence.
The SLC28 (CNT) and SLC29 (ENT) nucleoside transporter families: a 30-year collaborative odyssey
Authors: Young James D.
Published: June 2016

Specialized nucleoside transporter (NT) proteins are required for passage of nucleosides and hydrophilic nucleoside analogues across biological membranes. Physiologic nucleosides serve as central salvage metabolites in nucleotide biosynthesis, and nucleoside analogues are used as chemotherapeutic agents in the treatment of cancer and antiviral diseases. The nucleoside adenosine modulates numerous cellular events via purino-receptor cell signalling pathways. Human NTs are divided into two structurally unrelated protein families: the SLC28 concentrative nucleoside transporter (CNT) family and the SLC29 equilibrative nucleoside transporter (ENT) family. Human CNTs are inwardly directed Na+-dependent nucleoside transporters found predominantly in intestinal and renal epithelial and other specialized cell types. Human ENTs mediate bidirectional fluxes of purine and pyrimidine nucleosides down their concentration gradients and are ubiquitously found in most, possibly all, cell types. Both protein families are evolutionarily old: CNTs are present in both eukaryotes and prokaryotes; ENTs are widely distributed in mammalian, lower vertebrate and other eukaryote species. This mini-review describes a 30-year collaboration with Professor Stephen Baldwin to identify and understand the structures and functions of these physiologically and clinically important transport proteins.
Rotating with the brakes on and other unresolved features of the vacuolar ATPase
Authors: Rawson Shaun , Harrison Michael A. , Muench Stephen P.
Published: June 2016

The rotary ATPase family comprises the ATP synthase (F-ATPase), vacuolar ATPase (V-ATPase) and archaeal ATPase (A-ATPase). These either predominantly utilize a proton gradient for ATP synthesis or use ATP to produce a proton gradient, driving secondary transport and acidifying organelles. With advances in EM has come a significant increase in our understanding of the rotary ATPase family. Following the sub nm resolution reconstructions of both the F- and V-ATPases, the secondary structure organization of the elusive subunit a has now been resolved, revealing a novel helical arrangement. Despite these significant developments in our understanding of the rotary ATPases, there are still a number of unresolved questions about the mechanism, regulation and overall architecture, which this mini-review aims to highlight and discuss.
On the road to nowhere: cross-talk between post-translational protein targeting and cytosolic quality control
Authors: Casson Joseph , McKenna Michael , High Stephen
Published: June 2016

A well-defined co-translational pathway couples the synthesis and translocation of nascent polypeptides into and across the membrane of the endoplasmic reticulum (ER), thereby minimizing the possibility of the hydrophobic signals and transmembrane domains that such proteins contain from being exposed to the cytosol. Nevertheless, a proportion of these co-translational substrates may fail to reach the ER, and therefore mislocalize to the cytosol where their intrinsic hydrophobicity makes them aggregation-prone. A range of hydrophobic precursor proteins that employ alternative, post-translational, routes for ER translocation also contribute to the cytosolic pool of mislocalized proteins (MLPs). In this review, we detail how mammalian cells can efficiently deal with these MLPs by selectively targeting them for proteasomal degradation. Strikingly, this pathway for MLP degradation is regulated by cytosolic components that also facilitate the TRC40-dependent, post-translational, delivery of tail-anchored membrane proteins (TA proteins) to the ER. Among these components are small glutamine-rich tetratricopeptide repeat-containing protein α (SGTA) and Bcl-2-associated athanogene 6 (BAG6), which appear to play a decisive role in enforcing quality control over hydrophobic precursor proteins that have mislocalized to the cytosol, directing them to either productive membrane insertion or selective ubiquitination and proteasomal degradation.
Overcoming bottlenecks in the membrane protein structural biology pipeline
Authors: Hardy David , Bill Roslyn M. , Jawhari Anass , Rothnie Alice J.
Published: June 2016

Membrane proteins account for a third of the eukaryotic proteome, but are greatly under-represented in the Protein Data Bank. Unfortunately, recent technological advances in X-ray crystallography and EM cannot account for the poor solubility and stability of membrane protein samples. A limitation of conventional detergent-based methods is that detergent molecules destabilize membrane proteins, leading to their aggregation. The use of orthologues, mutants and fusion tags has helped improve protein stability, but at the expense of not working with the sequence of interest. Novel detergents such as glucose neopentyl glycol (GNG), maltose neopentyl glycol (MNG) and calixarene-based detergents can improve protein stability without compromising their solubilizing properties. Styrene maleic acid lipid particles (SMALPs) focus on retaining the native lipid bilayer of a membrane protein during purification and biophysical analysis. Overcoming bottlenecks in the membrane protein structural biology pipeline, primarily by maintaining protein stability, will facilitate the elucidation of many more membrane protein structures in the near future.
Construction of membrane-bound artificial cells using microfluidics: a new frontier in bottom-up synthetic biology
Authors: Elani Yuval
Published: June 2016

The quest to construct artificial cells from the bottom-up using simple building blocks has received much attention over recent decades and is one of the grand challenges in synthetic biology. Cell mimics that are encapsulated by lipid membranes are a particularly powerful class of artificial cells due to their biocompatibility and the ability to reconstitute biological machinery within them. One of the key obstacles in the field centres on the following: how can membrane-based artificial cells be generated in a controlled way and in high-throughput? In particular, how can they be constructed to have precisely defined parameters including size, biomolecular composition and spatial organization? Microfluidic generation strategies have proved instrumental in addressing these questions. This article will outline some of the major principles underpinning membrane-based artificial cells and their construction using microfluidics, and will detail some recent landmarks that have been achieved.
Lessons from making the Structural Classification of Proteins (SCOP) and their implications for protein structure modelling
Authors: Andreeva Antonina
Published: June 2016

The Structural Classification of Proteins (SCOP) database has facilitated the development of many tools and algorithms and it has been successfully used in protein structure prediction and large-scale genome annotations. During the development of SCOP, numerous exceptions were found to topological rules, along with complex evolutionary scenarios and peculiarities in proteins including the ability to fold into alternative structures. This article reviews cases of structural variations observed for individual proteins and among groups of homologues, knowledge of which is essential for protein structure modelling.
Artificial membranes for membrane protein purification, functionality and structure studies
Authors: Parmar Mayuriben J. , Lousa Carine De Marcos , Muench Stephen P. , Goldman Adrian , Postis Vincent L.G.
Published: June 2016

Membrane proteins represent one of the most important targets for pharmaceutical companies. Unfortunately, technical limitations have long been a major hindrance in our understanding of the function and structure of such proteins. Recent years have seen the refinement of classical approaches and the emergence of new technologies that have resulted in a significant step forward in the field of membrane protein research. This review summarizes some of the current techniques used for studying membrane proteins, with overall advantages and drawbacks for each method.
Sialic acid acquisition in bacteria–one substrate, many transporters
Authors: Thomas Gavin H.
Published: June 2016

The sialic acids are a family of 9-carbon sugar acids found predominantly on the cell-surface glycans of humans and other animals within the Deuterostomes and are also used in the biology of a wide range of bacteria that often live in association with these animals. For many bacteria sialic acids are simply a convenient source of food, whereas for some pathogens they are also used in immune evasion strategies. Many bacteria that use sialic acids derive them from the environment and so are dependent on sialic acid uptake. In this mini-review I will describe the discovery and characterization of bacterial sialic acids transporters, revealing that they have evolved multiple times across multiple diverse families of transporters, including the ATP-binding cassette (ABC), tripartite ATP-independent periplasmic (TRAP), major facilitator superfamily (MFS) and sodium solute symporter (SSS) transporter families. In addition there is evidence for protein-mediated transport of sialic acids across the outer membrane of Gram negative bacteria, which can be coupled to periplasmic processing of different sialic acids to the most common form, β-D-N-acetylneuraminic acid (Neu5Ac) that is most frequently taken up into the cell.
Crystallizing the function of the magnetosome membrane mineralization protein Mms6
Authors: Staniland Sarah S. , Rawlings Andrea E.
Published: June 2016

The literature on the magnetosome membrane (MM) protein, magnetosome membrane specific6 (Mms6), is reviewed. Mms6 is native to magnetotactic bacteria (MTB). These bacteria take up iron from solution and biomineralize magnetite nanoparticles within organelles called magnetosomes. Mms6 is a small protein embedded on the interior of the MM and was discovered tightly associated with the formed mineral. It has been the subject of intensive research as it is seen to control the formation of particles both in vivo and in vitro. Here, we compile, review and discuss the research detailing Mms6’s activity within the cell and in a range of chemical in vitro methods where Mms6 has a marked effect on the composition, size and distribution of synthetic particles, with approximately 21 nm in size for solution precipitations and approximately 90 nm for those formed on surfaces. Furthermore, we review and discuss recent work detailing the structure and function of Mms6. From the evidence, we propose a mechanism for its function as a specific magnetite nucleation protein and summaries the key features for this action: namely, self-assembly to display a charged surface for specific iron binding, with the curvature of the surfaces determining the particle size. We suggest these may aid design of biomimetic additives for future green nanoparticle production.
Towards developing algal synthetic biology
Authors: Scaife Mark Aden , Smith Alison Gail
Published: June 2016

The genetic, physiological and metabolic diversity of microalgae has driven fundamental research into photosynthesis, flagella structure and function, and eukaryotic evolution. Within the last 10 years these organisms have also been investigated as potential biotechnology platforms, for example to produce high value compounds such as long chain polyunsaturated fatty acids, pigments and antioxidants, and for biodiesel precursors, in particular triacylglycerols (TAGs). Transformation protocols, molecular tools and genome sequences are available for a number of model species including the green alga Chlamydomonas reinhardtii and the diatom Phaeodactylum tricornutum, although for both species there are bottlenecks to be overcome to allow rapid and predictable genetic manipulation. One approach to do this would be to apply the principles of synthetic biology to microalgae, namely the cycle of Design-Build-Test, which requires more robust, predictable and high throughput methods. In this mini-review we highlight recent progress in the areas of improving transgene expression, genome editing, identification and design of standard genetic elements (parts), and the use of microfluidics to increase throughput. We suggest that combining these approaches will provide the means to establish algal synthetic biology, and that application of standard parts and workflows will avoid parallel development and capitalize on lessons learned from other systems.