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Single Molecule
In situ mRNA isolation from a microfluidic single-cell array using an external AFM nanoprobe
Apr 12, 2017   Lab On A Chip
Li X, Tao Y, Lee DH, Wickramasinghe HK, Lee AP
In situ mRNA isolation from a microfluidic single-cell array using an external AFM nanoprobe
Apr 12, 2017
Lab On A Chip
We present an in situ mRNA extraction platform to quantify marker-genes' expression levels of single target cells within high-density microfluidic trapping arrays. This platform enables single-cell transcriptomic analysis to reveal in-depth information of cellular mechanisms and population heterogeneity. Although microfluidic technology enables the automation of single-cell sorting, trapping and identification, most developed microfluidic devices are closed off and prevent single-cell access by external analytical equipment. Besides, cell lysing is usually required for mRNA extraction. In our platform, cells are trapped individually in a microwell array sealed by a 1 μm-thick polydimethylsiloxane (PDMS) membrane, and a modified atomic force microscopy (AFM) probe-a dielectrophoretic nanotweezer (DENT)-penetrates through the membrane and extracts mRNA molecules from a single cell by dielectrophoresis. The single-cellular expression levels of 3 housekeeping genes from HeLa cells were analyzed quantitatively based on the quantification of the extracted mRNAs, and the probed cells remained viable when the applied alternating-current (AC) voltage was lower than 1.5 Vpp during mRNA probing. We also performed in situ mRNA isolation from a mixture of SK-BR-3 and U937 cells, mimicking a blood sample that underwent primary enrichment of circulating tumor cells (CTCs), and evaluated various marker-genes' expressions. This integrated platform combines the non-destructive and precise-control of a single-cell mRNA probe with sealed microfluidic systems' capability of upstream sample processing and downstream multifunctional analysis to enable a versatile and powerful tool for biomedical research.
Using microsecond single-molecule FRET to determine the assembly pathways of T4 ssDNA binding protein onto model DNA replication forks
Apr 18, 2017   Proceedings Of The National Academy Of Sciences Of The United States Of America
Phelps C, Israels B, Jose D, Marsh MC, von Hippel PH, Marcus AH
Using microsecond single-molecule FRET to determine the assembly pathways of T4 ssDNA binding protein onto model DNA replication forks
Apr 18, 2017
Proceedings Of The National Academy Of Sciences Of The United States Of America
DNA replication is a core biological process that occurs in prokaryotic cells at high speeds (∼1 nucleotide residue added per millisecond) and with high fidelity (fewer than one misincorporation event per 107 nucleotide additions). The ssDNA binding protein [gene product 32 (gp32)] of the T4 bacteriophage is a central integrating component of the replication complex that must continuously bind to and unbind from transiently exposed template strands during DNA synthesis. We here report microsecond single-molecule FRET (smFRET) measurements on Cy3/Cy5-labeled primer-template (p/t) DNA constructs in the presence of gp32. These measurements probe the distance between Cy3/Cy5 fluorophores that label the ends of a short (15-nt) segment of ssDNA attached to a model p/t DNA construct and permit us to track the stochastic interconversion between various protein bound and unbound states. The length of the 15-nt ssDNA lattice is sufficient to accommodate up to two cooperatively bound gp32 proteins in either of two positions. We apply a unique multipoint time correlation function analysis to the microsecond-resolved smFRET data obtained to determine and compare the kinetics of various possible reaction pathways for the assembly of cooperatively bound gp32 protein onto ssDNA sequences located at the replication fork. The results of our analysis reveal the presence and translocation mechanisms of short-lived intermediate bound states that are likely to play a critical role in the assembly mechanisms of ssDNA binding proteins at replication forks and other ss duplex junctions.
A monodisperse transmembrane α-helical peptide barrel
Apr 21, 2017   Nature Chemistry Add nature.com free-link Cancel
Mahendran KR, Niitsu A, Kong L, Thomson AR, Sessions RB, Woolfson DN, Bayley H
A monodisperse transmembrane α-helical peptide barrel
Apr 21, 2017
Nature Chemistry
The fabrication of monodisperse transmembrane barrels formed from short synthetic peptides has not been demonstrated previously. This is in part because of the complexity of the interactions between peptides and lipids within the hydrophobic environment of a membrane. Here we report the formation of a transmembrane pore through the self-assembly of 35 amino acid α-helical peptides. The design of the peptides is based on the C-terminal D4 domain of the Escherichia coli polysaccharide transporter Wza. By using single-channel current recording, we define discrete assembly intermediates and show that the pore is most probably a helix barrel that contains eight D4 peptides arranged in parallel. We also show that the peptide pore is functional and capable of conducting ions and binding blockers. Such α-helix barrels engineered from peptides could find applications in nanopore technologies such as single-molecule sensing and nucleic-acid sequencing.
Mineralization-inhibiting effects of transglutaminase-crosslinked polymeric osteopontin
Apr 21, 2017   Bone
Hoac B, Nelea V, Jiang W, Kaartinen MT, McKee MD
Mineralization-inhibiting effects of transglutaminase-crosslinked polymeric osteopontin
Apr 21, 2017
Bone
Osteopontin (OPN) belongs to the SIBLING family (Small, Integrin-Binding LIgand N-linked Glycoproteins) of mineral-binding matrix proteins found in bones and teeth. OPN is a well-known inhibitor of matrix mineralization, and enzymatic modification of OPN can affect this inhibitory function. In bone, OPN exists both as a monomer and as a high-molecular-weight polymer - the latter is formed by transglutaminase-mediated crosslinking of glutamine and lysine residues in OPN to create homotypic protein assemblies. OPN can be covalently crosslinked by transglutaminase 2 (TG2) and Factor XIII-A. Polymeric OPN has increased binding to collagen and promotes osteoblast adhesion, but despite these initial observations, its role in mineralization is not clear. In this study, we investigated the effect of polymerized OPN on mineralization using a hydroxyapatite crystal growth assay and mineralizing MC3T3-E1 osteoblast cultures. In the cultures, endogenous polymeric OPN was detected after mineralization occurred. In cell-free conditions, TG2 was used to crosslink bovine OPN into its polymeric form, and atomic force microscopy and dynamic light scattering revealed variably-sized, large branched aggregates ranging across hundreds of nanometers. These OPN polymers inhibited the growth of hydroxyapatite crystals in solution at concentrations similar to monomeric OPN, although the crosslinking slightly reduced its inhibitory potency. When added to MC3T3-E1 osteoblast cultures, this exogenous polymeric OPN essentially did not inhibit mineralization when given during the later mineralization stages of culture; however, cultures treated early and then continuously with polymeric OPN throughout both the matrix assembly and mineral deposition stages showed reduced mineralization. Immunoblotting of protein extracts from these continuously treated cultures revealed exogenous OPN polymers incorporated into mature matrix that had not yet mineralized. These results suggest that in bone, the increased size and branched structure of crosslinked inhibitory polymeric OPN near the mineralization front could hinder it from accessing focal mineralization sites in the dense collagen-rich matrix, suggesting that OPN-crosslinking into polymers may represent a way to fine-tune the inhibitory potency of OPN on bone mineralization. Copyright © 2016. Published by Elsevier Inc.
Plasma neurofilament light chain levels in Alzheimer's disease
Apr 21, 2017   Neuroscience Letters
Zhou W, Zhang J, Ye F, Xu G, Su H, Su Y, Zhang X, for Alzheimer’s Disease Neuroimaging Initiative
Plasma neurofilament light chain levels in Alzheimer's disease
Apr 21, 2017
Neuroscience Letters
Plasma neurofilament light (NFL) levels may be a marker of neuronal injury. We examined whether plasma NFL might be a potential biomarker for the prodromal and dementia stages of AD. Participants included 193 cognitively normal (CN), 198 amnestic mild cognitive impairment (aMCI) and 187 Alzheimer's disease (AD) individuals enrolled in the Alzheimer's Disease Neuroimaging Initiative (ADNI). Plasma NFL levels were examined by the Single Molecule array (Simoa) technique. Our results showed significantly increased plasma NFL levels in both AD (50.9pg/ml) and aMCI (43.0pg/ml) groups compared to CN (34.7pg/ml) group (both p
Ripping RNA by Force Using Gaussian Network Models
Apr 20, 2017   The Journal Of Physical Chemistry. B
Hyeon C, Thirumalai D
Ripping RNA by Force Using Gaussian Network Models
Apr 20, 2017
The Journal Of Physical Chemistry. B
Using force as a probe to map the folding landscapes of RNA molecules has become a reality thanks to major advances in single molecule pulling experiments. Although the unfolding pathways under tension are complicated to predict, studies in the context of proteins have shown that topology is the major determinant of the unfolding landscapes. By building on this finding we study the responses of RNA molecules to force by adapting Gaussian network model (GNM) that represents RNAs using a bead-spring network with isotropic interactions. Cross-correlation matrices of residue fluctuations, which are analytically calculated using GNM even upon application of mechanical force, show distinct allosteric communication as RNAs rupture. The model is used to calculate the force-extension curves at full thermodynamic equilibrium, and the corresponding unfolding pathways of four RNA molecules subject to a quasi-statically increased force. Our study finds that the analysis using GNM captures qualitatively the unfolding pathway of T. ribozyme elucidated by the optical tweezers measurement. However, the simple model cannot capture features, such as bifurcation in the unfolding pathways or the ion effects, in the forced-unfolding of RNAs.
Quantification of HER2 and estrogen receptor heterogeneity in breast cancer by single-molecule RNA fluorescence in situ hybridization
Apr 20, 2017   Oncotarget
Annaratone L, Simonetti M, Wernersson E, Marchiò C, Garnerone S, Scalzo MS, Bienko M, Chiarle R, Sapino A, Crosetto N
Quantification of HER2 and estrogen receptor heterogeneity in breast cancer by single-molecule RNA fluorescence in situ hybridization
Apr 20, 2017
Oncotarget
Intra-tumor heterogeneity is a pervasive property of human cancers that poses a major clinical challenge. Here, we describe the characterization, at the transcriptional level, of the intra-tumor topography of two prominent breast cancer biomarkers and drug targets, epidermal growth factor receptor 2 (HER2) and estrogen receptor 1 (ER) in 49 archival breast cancer samples. We developed a protocol for single-molecule RNA FISH in formalin-fixed, paraffin-embedded tissue sections (FFPE-smFISH), which enabled us to simultaneously detect and perform absolute quantification of HER2 and ER mature transcripts in single cells and multiple tumor regions. We benchmarked our method with standard diagnostic techniques, demonstrating that FFPE-smFISH is able to correctly classify breast cancers into well-established molecular subgroups. By counting transcripts in thousands of single cells, we identified different expression modes and levels of inter-cellular variability. In samples expressing both HER2 and ER, many cells co-expressed both genes, although expression levels were typically uncorrelated. Finally, we applied diversity metrics from the field of ecology to assess the intra-tumor topography of HER2 and ER gene expression, revealing that the spatial distribution of these key biomarkers can vary substantially even among breast cancers of the same subtype. Our results demonstrate that FFPE-smFISH is a reliable diagnostic assay and a powerful method for quantification of intra-tumor transcriptional heterogeneity of selected biomarkers in clinical samples.
Resolving Subcellular miRNA Trafficking and Turnover at Single-Molecule Resolution
Apr 19, 2017   Cell Reports
Pitchiaya S, Heinicke LA, Park JI, Cameron EL, Walter NG
Resolving Subcellular miRNA Trafficking and Turnover at Single-Molecule Resolution
Apr 19, 2017
Cell Reports
Regulation of microRNA (miRNA) localization and stability is critical for their extensive cytoplasmic RNA silencing activity and emerging nuclear functions. Here, we have developed single-molecule fluorescence-based tools to assess the subcellular trafficking, integrity, and activity of miRNAs. We find that seed-matched RNA targets protect miRNAs against degradation and enhance their nuclear retention. While target-stabilized, functional, cytoplasmic miRNAs reside in high-molecular-weight complexes, nuclear miRNAs, as well as cytoplasmic miRNAs targeted by complementary anti-miRNAs, are sequestered stably within significantly lower-molecular-weight complexes and rendered repression incompetent. miRNA stability and activity depend on Argonaute protein abundance, whereas miRNA strand selection, unwinding, and nuclear retention depend on Argonaute identity. Taken together, our results show that miRNA degradation competes with Argonaute loading and target binding to control subcellular miRNA abundance for gene silencing surveillance. Probing single cells for miRNA activity, trafficking, and metabolism promises to facilitate screening for effective miRNA mimics and anti-miRNA drugs. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.
3D single-molecule tracking enables direct hybridization kinetics measurement in solution
Apr 19, 2017   Nanoscale
Liu C, Obliosca JM, Liu YL, Chen YA, Jiang N, Yeh HC
3D single-molecule tracking enables direct hybridization kinetics measurement in solution
Apr 19, 2017
Nanoscale
Single-molecule measurements of DNA hybridization kinetics are mostly performed on a surface or inside a trap. Here we demonstrate a time-resolved, 3D single-molecule tracking (3D-SMT) method that allows us to follow a freely diffusing ssDNA molecule in solution for hundreds of milliseconds or even seconds and observe multiple annealing and melting events taking place on the same molecule. This is achieved by combining confocal-feedback 3D-SMT with time-domain fluorescence lifetime measurement, where fluorescence lifetime serves as the indicator of hybridization. With sub-diffraction-limit spatial resolution in molecular tracking and 15 ms temporal resolution in monitoring the change of reporter's lifetime, we have demonstrated a full characterization of annealing rate (kon = 5.13 × 106 M-1 s-1), melting rate (koff = 9.55 s-1), and association constant (Ka = 0.54 μM-1) of an 8 bp duplex model system diffusing at 4.8 μm2 s-1. As our method completely eliminates the photobleaching artifacts and diffusion interference, our kon and koff results well represent the real kinetics in solution. Our binding kinetics measurement can be carried out in a low signal-to-noise ratio condition (SNR ≈ 1.4) where ∼130 recorded photons are sufficient for a lifetime estimation. Using a population-level analysis, we can characterize hybridization kinetics over a wide range (0.5-125 s-1), even beyond the reciprocals of the lifetime monitoring temporal resolution and the average track duration.
Boundary layer friction of solvate ionic liquids as a function of potential
Apr 19, 2017   Faraday Discussions
Li H, Rutland MW, Watanabe M, Atkin R
Boundary layer friction of solvate ionic liquids as a function of potential
Apr 19, 2017
Faraday Discussions
Atomic force microscopy (AFM) has been used to investigate the potential dependent boundary layer friction at solvate ionic liquid (SIL)-highly ordered pyrolytic graphite (HOPG) and SIL-Au(111) interfaces. Friction trace and retrace loops of lithium tetraglyme bis(trifluoromethylsulfonyl)amide (Li(G4) TFSI) at HOPG present clearer stick-slip events at negative potentials than at positive potentials, indicating that a Li+ cation layer adsorbed to the HOPG lattice at negative potentials which enhances stick-slip events. The boundary layer friction data for Li(G4) TFSI shows that at HOPG, friction forces at all potentials are low. The TFSI- anion rich boundary layer at positive potentials is more lubricating than the Li+ cation rich boundary layer at negative potentials. These results suggest that boundary layers at all potentials are smooth and energy is predominantly dissipated via stick-slip events. In contrast, friction at Au(111) for Li(G4) TFSI is significantly higher at positive potentials than at negative potentials, which is comparable to that at HOPG at the same potential. The similarity of boundary layer friction at negatively charged HOPG and Au(111) surfaces indicates that the boundary layer compositions are similar and rich in Li+ cations for both surfaces at negative potentials. However, at Au(111), the TFSI- rich boundary layer is less lubricating than the Li+ rich boundary layer, which implies that anion reorientations rather than stick-slip events are the predominant energy dissipation pathways. This is confirmed by the boundary friction of Li(G4) NO3 at Au(111), which shows similar friction to Li(G4) TFSI at negative potentials due to the same cation rich boundary layer composition, but even higher friction at positive potentials, due to higher energy dissipation in the NO3- rich boundary layer.
Detecting Single-Nucleotides by Tunneling Current Measurements at Sub-MHz Temporal Resolution
Apr 19, 2017   Sensors (Basel, Switzerland)
Morikawa T, Yokota K, Tanimoto S, Tsutsui M, Taniguchi M
Detecting Single-Nucleotides by Tunneling Current Measurements at Sub-MHz Temporal Resolution
Apr 19, 2017
Sensors (Basel, Switzerland)
Label-free detection of single-nucleotides was performed by fast tunneling current measurements in a polar solvent at 1 MHz sampling rate using SiO₂-protected Au nanoprobes. Short current spikes were observed, suggestive of trapping/detrapping of individual nucleotides between the nanoelectrodes. The fall and rise features of the electrical signatures indicated signal retardation by capacitance effects with a time constant of about 10 microseconds. The high temporal resolution revealed current fluctuations, reflecting the molecular conformation degrees of freedom in the electrode gap. The method presented in this work may enable direct characterizations of dynamic changes in single-molecule conformations in an electrode gap in liquid.
CBMs as Probes to Explore Plant Cell Wall Heterogeneity Using Immunocytochemistry
Apr 18, 2017   Methods In Molecular Biology (Clifton, N.J.)
Badruna L, Burlat V, Montanier CY
CBMs as Probes to Explore Plant Cell Wall Heterogeneity Using Immunocytochemistry
Apr 18, 2017
Methods In Molecular Biology (Clifton, N.J.)
Immunocytochemistry is a widely used technique to localize antigen within intact tissues. Plant cell walls are complex matrixes of highly decorated polysaccharides and the large number of CBM families displaying specific substrate recognition reflects this complexity. The accessibility of large proteins, such as antibodies, to their cell wall epitopes may be sometimes difficult due to steric hindrance problems. Due to their smaller size, CBMs are interesting alternative probes. The aim of this chapter is to describe the use of CBM as probes to explore complex polysaccharide topochemistry in muro and to quantify enzymatic deconstruction.
p53 dynamically directs TFIID assembly on target gene promoters
Apr 18, 2017   Molecular And Cellular Biology
Coleman RA, Qiao Z, Singh SK, Peng CS, Cianfrocco M, Zhang Z, Piasecka A, Aldeborgh H, Basishvili G, Liu WL
p53 dynamically directs TFIID assembly on target gene promoters
Apr 18, 2017
Molecular And Cellular Biology
p53 is a central regulator that turns on vast gene networks to maintain cellular integrity upon various stimuli. p53 activates transcription initiation in part by aiding recruitment of TFIID to the promoter. However, the precise means by which p53 dynamically interacts with TFIID to facilitate assembly on target gene promoters remains elusive. To address this key question, we have undertaken an integrated approach involving single molecule fluorescence microscopy, single particle cryo-electron microscopy, and biochemistry. Our real-time single molecule imaging demonstrates that TFIID alone binds poorly to native p53 target promoters. p53 unlocks TFIID's ability to bind DNA by stabilizing TFIID contacts with both the core promoter and a region within p53's response element. Analysis of single molecule dissociation kinetics reveals that TFIID interacts with promoters via transient and prolonged DNA binding modes that are each regulated by p53. Importantly, our structural work reveals that TFIID's conversion to a rearranged DNA-binding conformation is enhanced in the presence of DNA and p53. Notably, TFIID's interaction with DNA induces p53 to rapidly dissociate, which likely leads to additional rounds of p53-mediated recruitment of other basal factors. Collectively, these findings indicate that p53 dynamically escorts and loads TFIID onto its target promoters. Copyright © 2017 American Society for Microbiology.
Nanomechanics of individual aerographite tetrapods
Apr 12, 2017   Nature Communications
Meija R, Signetti S, Schuchardt A, Meurisch K, Smazna D, Mecklenburg M, Schulte K, Erts D, Lupan O, Fiedler B, Mishra YK, Adelung R, Pugno NM
Nanomechanics of individual aerographite tetrapods
Apr 12, 2017
Nature Communications
Carbon-based three-dimensional aerographite networks, built from interconnected hollow tubular tetrapods of multilayer graphene, are ultra-lightweight materials recently discovered and ideal for advanced multifunctional applications. In order to predict the bulk mechanical behaviour of networks it is very important to understand the mechanics of their individual building blocks. Here we characterize the mechanical response of single aerographite tetrapods via in situ scanning electron and atomic force microscopy measurements. To understand the acquired results, which show that the overall behaviour of the tetrapod is governed by the buckling of the central joint, a mechanical nonlinear model was developed, introducing the concept of the buckling hinge. Finite element method simulations elucidate the governing buckling phenomena. The results are then generalized for tetrapods of different size-scales and shapes. These basic findings will permit better understanding of the mechanical response of the related networks and the design of similar aerogels based on graphene and other two-dimensional materials.
UV-assisted treatment on hydrophobic acrylic IOLs anterior surface with methacryloyloxyethyl phosphorylcholine: Reducing inflammation and maintaining low posterior capsular opacification properties
Apr 18, 2017   Materials Science & Engineering. C, Materials For Biological Applications
Huang X, Luo C, Lin L, Zhang L, Li H, Yao K, Xu Z
UV-assisted treatment on hydrophobic acrylic IOLs anterior surface with methacryloyloxyethyl phosphorylcholine: Reducing inflammation and maintaining low posterior capsular opacification properties
Apr 18, 2017
Materials Science & Engineering. C, Materials For Biological Applications
In order to enhance the surface biocompatibility of hydrophobic acrylic intraocular lenses (IOLs), the anterior surface was modified through graft polymerization with methacryloyloxyethyl phosphorylcholine (MPC) initiated by ultraviolet radiation. Chemical changes on the IOL surface were characterised by X-ray photoelectron spectroscopy (XPS) to confirm the covalent binding of MPC. Field emission scanning electron microscopy (FESEM) and atomic force microscopy (AFM) analysis were used to characterise the morphological changes to the IOL surface. Static water contact angle (WCA) measurements showed that the hydrophilicity of the anterior surface was enhanced after grafting with MPC. The surface biocompatibility of the untreated and Anterior UV-MPC IOLs was compared with the adhesion behaviour of macrophages in vitro. The degree of posterior capsule opacification (PCO) of untreated, anterior treated, and posterior treated IOLs was evaluated by observing the behaviour of lens epithelial cells (LECs) between the IOL optic portion and type IV collagen. Results suggest that the MPC modified anterior surface resisted the attachment of macrophages. However, MPC grafted onto the posterior surface enhanced the migration of LECs between the IOL optic and type IV collagen, which may result in severe PCO. In conclusion, our Anterior UV-MPC IOLs may reduce postoperative inflammation while maintaining the low PCO rates of hydrophobic acrylic IOLs. Copyright © 2017 Elsevier B.V. All rights reserved.
An orthogonal single-molecule experiment reveals multiple-attempt dynamics of type IA topoisomerases
Apr 17, 2017   Nature Structural & Molecular Biology Add nature.com free-link Cancel
Gunn KH, Marko JF, Mondragón A
An orthogonal single-molecule experiment reveals multiple-attempt dynamics of type IA topoisomerases
Apr 17, 2017
Nature Structural & Molecular Biology
Topoisomerases are enzymes that are involved in maintaining the topological state of cellular DNA. Their dynamic characteristics remain poorly understood despite numerous structural, biophysical and biochemical studies. Recent single-molecule experiments revealed that an important feature of the type IA topoisomerase mechanism is the presence of pauses between relaxation events. However, these experiments could not determine whether the protein remains DNA bound during the pauses or what relationship may exist between protein domain movements and topological changes in the DNA. By combining two orthogonal single-molecule techniques, we found that E. coli topoisomerase I constantly changes conformation when attempting to modify the topology of DNA, but succeeds in only a fraction of the attempts. Thus, its mechanism can be described as a series of DNA strand-passage attempts that culminate in a successful relaxation event.
Single-molecule and super-resolution imaging of transcription in living bacteria
Apr 17, 2017   Methods (San Diego, Calif.)
Stracy M, Kapanidis AN
Single-molecule and super-resolution imaging of transcription in living bacteria
Apr 17, 2017
Methods (San Diego, Calif.)
In vivo single-molecule and super-resolution techniques are transforming our ability to study transcription as it takes place in its native environment in living cells. This review will detail the methods for imaging single molecules in cells, and the data-analysis tools which can be used to extract quantitative information on the spatial organization, mobility, and kinetics of the transcription machinery from these experiments. Furthermore, we will highlight studies which have applied these techniques to shed new light on bacterial transcription. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.
Escherichia coli adhesive coating as a chiral stationary phase for open tubular capillary electrochromatography enantioseparation
Apr 16, 2017   Analytica Chimica Acta
Fu Q, Zhang K, Gao D, Wang L, Yang F, Liu Y, Xia Z
Escherichia coli adhesive coating as a chiral stationary phase for open tubular capillary electrochromatography enantioseparation
Apr 16, 2017
Analytica Chimica Acta
Bacteria, the microorganism with intrinsic chirality, have numerous fascinating chiral phenomena such as various chirality-triggered biological processes and behaviors. Herein, bacteria were firstly explored as novel chiral stationary phases in open-tubular capillary electrochromatography (OT-CEC) for enantioseparation of fluoroquinolone enantiomers and simultaneous separation of six fluoroquinolone antibiotics. The model strain, i.e. non-pathogenic Escherichia coli (E. coli) DH5α, was adhered onto the inner surface of positively charged polyethyleneimine (PEI) modified capillaries based on the bacterial adhesion characteristics and strong electrostatic interaction. The morphology and thickness of the bacteria adhesive coatings in the capillary were characterized by field emission scanning electron microscopy (FESEM) and atomic force microscopy (AFM). Baseline separation of ofloxacin and partial separation of lomefloxacin enantiomers could be achieved by the E. coli coated columns. The preparation parameters including the coating time and concentration of bacteria that affecting the chiral resolution were intensively investigated. The electrophoretic parameters, including pH, buffer concentration and applied voltage, were also optimized. The developed method was validated (linearity, LOD, LOQ, intra-day, inter-day and column-to-column repeatability and recovery) and successfully utilized for the quantitative analysis of ofloxacin enantiomers in the ofloxacin tablets. Moreover, only a slight decrease in the separation efficiency was observed after 90 consecutive runs on the E. coli@capillary. These results demonstrated that bacteria are promising stationary phases for chiral separation in CEC. Copyright © 2017 Elsevier B.V. All rights reserved.
PLGA-PEG nano-delivery system for epigenetic therapy
Apr 13, 2017   Biomedicine & Pharmacotherapy = Biomedecine & Pharmacotherapie
Naz A, Cui Y, Collins CJ, Thompson DH, Irudayaraj J
PLGA-PEG nano-delivery system for epigenetic therapy
Apr 13, 2017
Biomedicine & Pharmacotherapy = Biomedecine & Pharmacotherapie
Efficient delivery of cytidine analogues such as Azacitidine (AZA) into solid tumors constitutes a primary challenge in epigenetic therapies. We developed a di-block nano-vector based on poly(lactic-co-glycolic acid) (PLGA) and poly(ethylene glycol) (PEG) for stabilization of the conjugated AZA under physiological conditions. With equimolar drug content, our nano-conjugate could elicit a better anti-proliferative effect over free drug in breast cancer both in vitro and in vivo, through reactivation of p21 and BRCA1 to restrict cell proliferation. In addition, we applied single-molecule fluorescence tools to characterize the intracellular behavior of the AZA-PLGE-PEG nano-micelles at a finer spatiotemporal resolution. Our results suggest that the nano-micelles could effectively enrich in cancer cells and may not be limited by nucleoside transporters. Afterwards, the internalized nano-micelles exhibit pH-dependent release and resistance to active efflux. Altogether, our work describes a delivery strategy for DNA demethylating agents with nanoscale tunability, providing a cost-effective option for pharmaceutics. Copyright © 2017 Elsevier Masson SAS. All rights reserved.
Proteogenomic analysis reveals alternative splicing and translation as part of the abscisic acid response in Arabidopsis seedlings
Apr 13, 2017   The Plant Journal : For Cell And Molecular Biology
Zhu FY, Chen MX, Ye NH, Shi L, Ma KL,   . . . . . .   , Zhang D, Hao GF, Xiao S, Liu YG, Zhang J
Proteogenomic analysis reveals alternative splicing and translation as part of the abscisic acid response in Arabidopsis seedlings
Apr 13, 2017
The Plant Journal : For Cell And Molecular Biology
In eukaryotes, mechanisms such as alternative splicing (AS) and alternative translation initiation (ATI) contribute to organismal protein diversity. Specifically, splicing factors play crucial roles in responses to environment and development cues. However, the underlying mechanisms are not well investigated in plants. Here, we report the parallel employment of short-read RNA sequencing, single molecule long-read sequencing and proteomic identification to unravel AS isoforms and previously unannotated proteins in response to abscisic acid (ABA) treatment. Combining the data of the two sequencing methods, approximately 83.4% of intron-containing genes were alternatively spliced. Two AS types, which are referred to as alternative first exon (AFE) and alternative last exon (ALE), were more abundant than intron retention (IR). However, by contrast to AS events detected under normal conditions, differentially expressed AS isoforms were more likely to be translated. ABA extensively affects the AS pattern indicated by increasing number of non-conventional splicing sites. This work also identified thousands of unannotated peptides and proteins by ATI based on mass spectrometry and a virtual peptide library deduced from both strands of coding regions within the Arabidopsis genome. The results enhance our understanding on AS and alternative translation mechanisms under normal condition and in response to ABA treatment. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
Single-molecule electrical contacts on silicon electrodes under ambient conditions
Apr 13, 2017   Nature Communications
Aragonès AC, Darwish N, Ciampi S, Sanz F, Gooding JJ, Díez-Pérez I
Single-molecule electrical contacts on silicon electrodes under ambient conditions
Apr 13, 2017
Nature Communications
The ultimate goal in molecular electronics is to use individual molecules as the active electronic component of a real-world sturdy device. For this concept to become reality, it will require the field of single-molecule electronics to shift towards the semiconducting platform of the current microelectronics industry. Here, we report silicon-based single-molecule contacts that are mechanically and electrically stable under ambient conditions. The single-molecule contacts are prepared on silicon electrodes using the scanning tunnelling microscopy break-junction approach using a top metallic probe. The molecular wires show remarkable current-voltage reproducibility, as compared to an open silicon/nano-gap/metal junction, with current rectification ratios exceeding 4,000 when a low-doped silicon is used. The extension of the single-molecule junction approach to a silicon substrate contributes to the next level of miniaturization of electronic components and it is anticipated it will pave the way to a new class of robust single-molecule circuits.
Serum neurofilament light as a biomarker for mild traumatic brain injury in contact sports
Apr 13, 2017   Neurology
Shahim P, Zetterberg H, Tegner Y, Blennow K
Serum neurofilament light as a biomarker for mild traumatic brain injury in contact sports
Apr 13, 2017
Neurology
To evaluate whether the axonal protein neurofilament light (NFL) in serum is a sensitive biomarker to detect subtle brain injury or concussion in contact sports athletes. Two prospective cohort studies involving (1) 14 Swedish amateur boxers who underwent fluid biomarker assessments at 7-10 days after bout and after 3 months of rest from boxing and (2) 35 Swedish professional hockey players who underwent blood biomarker assessment at 1, 12, 36, and 144 hours after concussion and when the players returned to play were performed. Fourteen healthy nonathletic controls and 12 athletic controls were also enrolled. Serum NFL was measured using ultrasensitive single molecule array technology. Serum NFL concentrations were increased in boxers 7-10 days after bout as compared to the levels after 3 months rest as well as compared with controls (p = 0.0007 and p < 0.0001, respectively). NFL decreased following 3 months of rest, but was still higher than in controls (p < 0.0001). Boxers who received many (>15) hits to the head or were groggy after bout had higher concentrations of serum NFL as compared to those who received fewer hits to the head (p = 0.0023). Serum NFL increased over time in hockey players, and the levels returned to normal at return to play. Importantly, serum NFL could separate players with rapidly resolving postconcussion symptoms (PCS) from those with prolonged PCS. The results from these 2 independent cohort studies suggest that serum NFL is a highly sensitive biomarker for concussion. © 2017 American Academy of Neurology.
Binding of interferon reduces the force of unfolding for interferon receptor 1
Apr 13, 2017   PloS One
Chuartzman SG, Nevo R, Waichman S, Shental D, Piehler J, Levy Y, Reich Z, Kapon R
Binding of interferon reduces the force of unfolding for interferon receptor 1
Apr 13, 2017
PloS One
Differential signaling of the type I interferon receptor (IFNAR) has been correlated with the ability of its subunit, IFNAR1, to differentially recognize a large spectrum of different ligands, which involves intricate conformational re-arrangements of multiple interacting domains. To shed light onto the structural determinants governing ligand recognition, we compared the force-induced unfolding of the IFNAR1 ectodomain when bound to interferon and when free, using the atomic force microscope and steered molecular dynamics simulations. Unexpectedly, we find that IFNAR1 is easier to mechanically unfold when bound to interferon than when free. Analysis of the structures indicated that the origin of the reduction in unfolding forces is a conformational change in IFNAR1 induced by ligand binding.
Three-Dimensional Super-Resolution in Eukaryotic Cells Using the Double-Helix Point Spread Function
Apr 13, 2017   Biophysical Journal
Carr AR, Ponjavic A, Basu S, McColl J, Santos AM, Davis S, Laue ED, Klenerman D, Lee SF
Three-Dimensional Super-Resolution in Eukaryotic Cells Using the Double-Helix Point Spread Function
Apr 13, 2017
Biophysical Journal
Single-molecule localization microscopy, typically based on total internal reflection illumination, has taken our understanding of protein organization and dynamics in cells beyond the diffraction limit. However, biological systems exist in a complicated three-dimensional environment, which has required the development of new techniques, including the double-helix point spread function (DHPSF), to accurately visualize biological processes. The application of the DHPSF approach has so far been limited to the study of relatively small prokaryotic cells. By matching the refractive index of the objective lens immersion liquid to that of the sample media, we demonstrate DHPSF imaging of up to 15-μm-thick whole eukaryotic cell volumes in three to five imaging planes. We illustrate the capabilities of the DHPSF by exploring large-scale membrane reorganization in human T cells after receptor triggering, and by using single-particle tracking to image several mammalian proteins, including membrane, cytoplasmic, and nuclear proteins in T cells and embryonic stem cells. Crown Copyright © 2017. Published by Elsevier Inc. All rights reserved.
Single-molecule visualization of fast polymerase turnover in the bacterial replisome
Apr 22, 2017   ELife
Lewis JS, Spenkelink LM, Jergic S, Wood EA, Monachino E, Horan NP, Duderstadt KE, Cox MM, Robinson A, Dixon NE, van Oijen AM
Single-molecule visualization of fast polymerase turnover in the bacterial replisome
Apr 22, 2017
ELife
The Escherichia coli DNA replication machinery has been used as a road map to uncover design rules that enable DNA duplication with high efficiency and fidelity. Although the enzymatic activities of the replicative DNA Pol III are well understood, its dynamics within the replisome are not. Here we test the accepted view that the Pol III holoenzyme remains stably associated within the replisome. We use in vitro single-molecule assays with fluorescently labeled polymerases to demonstrate that the Pol III* complex (holoenzyme lacking the β2 sliding clamp), is rapidly exchanged during processive DNA replication. Nevertheless, the replisome is highly resistant to dilution in the absence of Pol III* in solution. We further show similar exchange in live cells containing labeled clamp loader and polymerase. These observations suggest a concentration-dependent exchange mechanism providing a balance between stability and plasticity, facilitating replacement of replisomal components dependent on their availability in the environment.

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