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1.  The interfacial structure and Young's modulus of peptide films having switchable mechanical properties 
We report the structure and Young's modulus of switchable films formed by peptide self-assembly at the air–water interface. Peptide surfactant AM1 forms an interfacial film that can be switched, reversibly, from a high- to low-elasticity state, with rapid loss of emulsion and foam stability. Using neutron reflectometry, we find that the AM1 film comprises a thin (approx. 15 Å) layer of ordered peptide in both states, confirming that it is possible to drastically alter the mechanical properties of an interfacial ensemble without significantly altering its concentration or macromolecular organization. We also report the first experimentally determined Young's modulus of a peptide film self-assembled at the air–water interface (E=80 MPa for AM1, switching to E<20 MPa). These findings suggest a fundamental link between E and the macroscopic stability of peptide-containing foam. Finally, we report studies of a designed peptide surfactant, Lac21E, which we find forms a stronger switchable film than AM1 (E=335 MPa switching to E<4 MPa). In contrast to AM1, Lac21E switching is caused by peptide dissociation from the interface (i.e. by self-disassembly). This research confirms that small changes in molecular design can lead to similar macroscopic behaviour via surprisingly different mechanisms.
doi:10.1098/rsif.2007.1063
PMCID: PMC2605502  PMID: 17550885
peptide; Young's modulus; interface; film; neutron reflectometry
2.  Photoswitchable gel assembly based on molecular recognition 
Nature Communications  2012;3:603-.
The formation of effective and precise linkages in bottom-up or top-down processes is important for the development of self-assembled materials. Self-assembly through molecular recognition events is a powerful tool for producing functionalized materials. Photoresponsive molecular recognition systems can permit the creation of photoregulated self-assembled macroscopic objects. Here we demonstrate that macroscopic gel assembly can be highly regulated through photoisomerization of an azobenzene moiety that interacts differently with two host molecules. A photoregulated gel assembly system is developed using polyacrylamide-based hydrogels functionalized with azobenzene (guest) or cyclodextrin (host) moieties. Reversible adhesion and dissociation of the host gel from the guest gel may be controlled by photoirradiation. The differential affinities of α-cyclodextrin or β-cyclodextrin for the trans-azobenzene and cis-azobenzene are employed in the construction of a photoswitchable gel assembly system.
Self-assembly through molecular recognition events is used in the production of functionalized materials. This study shows that macroscopic gel assembly can be regulated through photoisomerization of an azobenzene moiety that interacts differently with two host molecules.
doi:10.1038/ncomms1617
PMCID: PMC3272571  PMID: 22215078
3.  Photon-Regulated DNA-Enzymatic Nanostructures by Molecular Assembly 
ACS nano  2011;5(12):10090-10095.
Future smart nanostructures will have to rely on molecular assembly for unique or advanced desired functions. For example, the evolved ribosome in nature is one example of functional self-assembly of nucleic acids and proteins employed in nature to perform specific tasks. Artificial self-assembled nanodevices have also been developed to mimic key biofunctions, and various nucleic acid- and protein-based functional nanoassemblies have been reported. However, functionally regulating these nanostructures is still a major challenge. Here we report a general approach to fine-tune the catalytic function of DNA-enzymatic nanosized assemblies by taking advantage of the trans-cis isomerization of azobenzene molecules. To the best of our knowledge, this is the first study to precisely modulate the structures and functions of an enzymatic assembly based on light-induced DNA scaffold switching. Via photocontrolled DNA conformational switching, the proximity of multiple enzyme catalytic centers can be adjusted, as well as the catalytic efficiency of cofactor-mediated DNAzymes. We expect that this approach will lead to the advancement of DNA-enzymatic functional nanostructures in future biomedical and analytical applications.
doi:10.1021/nn204007y
PMCID: PMC3246559  PMID: 22098552
azobenzene; DNA-enzyme conjugation; enzyme cascade; photo controllable
4.  4-{(E)-2-[4-(But-3-en-1-yl­oxy)phen­yl]diazen-1-yl}benzoic acid 
The title compound, C17H16N2O3, has an E conformation about the azobenzene (–N=N–) linkage. The benzene rings are twisted slightly with respect to each other [6.79 (9)°], while the dihedral angle between the plane through the carb­oxy group and the attached benzene ring is 3.2 (2)°. In the crystal, mol­ecules are oriented with the carb­oxy groups head-to-head, forming O—H⋯O hydrogen-bonded inversion dimers. These dimers are connected by C—H⋯O hydrogen-bonds into layers lying parallel to the (013) plane.
doi:10.1107/S1600536812038718
PMCID: PMC3470328  PMID: 23125741
5.  Reversible structural switching of a DNA-DDAB film 
We describe the novel structure and behavior of a DNA-DDAB complex film cast from an organic solvent which exhibits a structural switching transition as it is dried or wetted with water. The film can be easily prepared by forming a complex between the negatively charged phosphate groups of DNA and the positively charged headgroup of the surfactant DDAB. This complex is then purified, dried, dissolved in isopropanol and cast onto a glass slide to form a self-standing film by means of slow evaporation. While the structure of the dried film was found to be composed of single-stranded DNA and a monolayer of DDAB, upon hydration of the film the structure switched to double stranded DNA complexed to a bilayer of DDAB. We expect that this phenomenon would serve as a useful model for the design of new responsive materials and programmable self-assembly.
doi:10.1021/ja809349m
PMCID: PMC2664520  PMID: 19275252
6.  Hierarchical flexural strength of enamel: transition from brittle to damage-tolerant behaviour 
Hard, biological materials are generally hierarchically structured from the nano- to the macro-scale in a somewhat self-similar manner consisting of mineral units surrounded by a soft protein shell. Considerable efforts are underway to mimic such materials because of their structurally optimized mechanical functionality of being hard and stiff as well as damage-tolerant. However, it is unclear how different hierarchical levels interact to achieve this performance. In this study, we consider dental enamel as a representative, biological hierarchical structure and determine its flexural strength and elastic modulus at three levels of hierarchy using focused ion beam (FIB) prepared cantilevers of micrometre size. The results are compared and analysed using a theoretical model proposed by Jäger and Fratzl and developed by Gao and co-workers. Both properties decrease with increasing hierarchical dimension along with a switch in mechanical behaviour from linear-elastic to elastic-inelastic. We found Gao's model matched the results very well.
doi:10.1098/rsif.2011.0498
PMCID: PMC3350718  PMID: 22031729
hierarchical structures; mechanical properties; biological materials; dental enamel
7.  Manipulation and assembly of small objects in liquid crystals by dynamical disorganizing effect of push-pull-azobenzene-dye 
Scientific Reports  2013;3:2167.
The phase transition of a nematic liquid crystal containing a push-pull azobenzene dye could be induced efficiently during irradiation with visible light. The dynamical disorganizing effect of the push-pull azobenzene dye on the liquid crystalline order through its trans-cis-trans photoisomerizaion cycle under visible light was contributed to the efficient phase transition. Then, the effects of light irradiation on the motion of small objects dispersed in the liquid crystals containing the push-pull azobenzene were explored, and the manipulation and assembly of those objects were successfully achieved in the nematic phase but also in the smectic phase. The combination of the photo-controlled dynamical change in the liquid crystalline order and the intrinsic self-assembly property of a liquid crystal is promising for use in technologies that require not only the organization of small objects but also the photo-driving of nano- and micro-sized mechanical materials.
doi:10.1038/srep02167
PMCID: PMC3705281  PMID: 23835605
8.  Mechanical characterization of carbon nanomembranes from self-assembled monolayers 
Summary
This paper reports on the mechanical characterization of carbon nanomembranes (CNMs) with a thickness of 1 nm that are fabricated by electron-induced crosslinking of aromatic self-assembled monolayers (SAMs). A novel type of in situ bulge test employing an atomic force microscope (AFM) is utilized to investigate their mechanical properties. A series of biphenyl-based molecules with different types of terminal and/or anchor groups were used to prepare the CNMs, such as 4'-[(3-trimethoxysilyl)propoxy]-[1,1'-biphenyl]-4-carbonitrile (CBPS), 1,1'-biphenyl-4-thiol (BPT) and 4-nitro-1,1'-biphenyl-4-thiol (NBPT). The elastic properties, viscoelastic behaviors and ultimate tensile strength of these biphenyl-based CNMs are investigated and discussed.
doi:10.3762/bjnano.2.92
PMCID: PMC3257509  PMID: 22259767
bulge test; carbon nanomembrane; mechanical characterization; self-assembled monolayers; two-dimensional materials
9.  Building a Nanostructure with Reversible Motions Using Photonic Energy 
ACS nano  2012;6(9):7935-7941.
Recently, the specific hybridization of DNA molecules has been used to construct self-assembled devices, e.g., the mechanical device to mimic cellular protein motors in nature. Here, we present a new light-powered DNA mechanical device based on the photoisomerization of azobenzene moieties and toehold-mediated strand displacement. This autonomous and controllable device is capable of moving towards either end of the track, simply by switching the wavelength of light irradiation, either UV (365nm) or visible (>450nm). This light-controlled strategy can easily solve one main technical challenge for step-wise walking devices: the selection of routes in multipath systems. The principle employed in this study, photoisomerization-induced toehold length switching, could be further useful in the design of other mechanical devices, with the ultimate goal of rivaling molecular motors for cargo transport and macroscopic movement.
doi:10.1021/nn302388e
PMCID: PMC3458132  PMID: 22823186
DNA nanostructure; light control; reversible motion; strand displacement
10.  (E)-1-[2-(2-Cyano­phen­yl)diazen-2-ium-1-yl]naphthalen-2-olate 
There are two independent zwitterion mol­ecules (A and B) in the asymmetric unit of the title compound, C17H11N3O, which belongs to the family of azo dyes. The dihedral angle between the benzene ring and the naphthalene ring system is 6.99 (6)° in mol­ecule A and 4.38 (6)° in mol­ecule B. The azo group adopts an E conformation with respect to the –N=N– bond and each of the independent mol­ecules has an intra­molecular N—H⋯O hydrogen bond. In the crystal, mol­ecules are linked by C—H⋯O and C—H⋯N hydrogen bonds, forming ribbons propagating along [-110]. The ribbons are linked via π–π inter­actions involving the benzene and naphthalene rings of inversion-related A and inversion-related B mol­ecules, forming a three-dimensional structure. The most significant centroid–centroid distances vary from 3.6599 (6) to 3.7538 (9) Å.
doi:10.1107/S1600536813017261
PMCID: PMC3770433  PMID: 24046718
11.  Photon-Manipulated Drug Release from Mesoporous Nanocontainer Controlled by Azobenzene-Modified Nucleic Acid 
ACS nano  2012;6(7):6337-6344.
Herein a photon manipulated mesoporous release system was constructed based on azobenzene-modified nucleic acids. In this system, the azobenzene-incorporated DNA double strands were immobilized at the pore mouth of meso-porous silica nanoparticles. The photo-isomerization of azobenzene induced dehybridization/hybridization switch of complementary DNA, causing uncapping/capping of pore gates of mesoporous silica. This nanoplatform permits holding of guest molecules within the nanopores under visible light but release them when light wavelength turns to UV range. These DNA/mesoporous silica hybrid nanostructures were exploited as carriers for cancer cell chemotherapy drug doxorubicin (DOX) due to its stimuli-responsive property as well as good biocompatibility via MTT assay. It is found that the drug release behavior is light wavelength sensitive. Switching of the light from visible to UV range uncapped the pores causes the release of DOX from the mesoporous silica nanospheres and an obvious cytotoxic effect on cancer cells. We envision that this photo-controlled drug release system could find potential applications in cancer therapy.
doi:10.1021/nn3018365
PMCID: PMC3407578  PMID: 22670595
azobenzene; photoregulation; mesoporous silica; nucleic acids; drug delivery
12.  Generating and measuring the anisotropic elastic behaviour of Co thin films with oriented surface nano-strings on micro-cantilevers 
Nanoscale Research Letters  2011;6(1):325.
In this research, the elastic behaviour of two Co thin films simultaneously deposited in an off-normal angle method was studied. Towards this end, two Si micro-cantilevers were simultaneously coated using pulsed laser deposition at an oblique angle, creating a Co nano-string surface morphology with a predetermined orientation. The selected position of each micro-cantilever during the coating process created longitudinal or transverse nano-strings. The anisotropic elastic behaviour of these Co films was determined by measuring the changes that took place in the resonant frequency of each micro-cantilever after this process of creating differently oriented plasma coatings had been completed. This differential procedure allowed us to determine the difference between the Young's modulus of the different films based on the different direction of the nano-strings. This difference was determined to be, at least, the 20% of the Young's modulus of the bulk Co.
PACS: 62.25.-g; 81.16.Rf; 68.60.Bs; 81.15.Fg; 68.37.Ef; 85.85.+j
doi:10.1186/1556-276X-6-325
PMCID: PMC3211413  PMID: 21711815
13.  1-[(E)-4-(Phenyl­diazen­yl)phen­yl]-3-pyrroline-2,5-dione 
The title compound, C16H11N3O2, displays a trans configuration with respect to the azo group. The mol­ecule is non-planar; the maleimide ring forms a dihedral angle of 42.35 (4)° with the benzene ring bonded to its N atom and the mean plane of this benzene ring is rotated by 21.46 (8)° with respect to the azo group mean plane, which, in turn, forms a dihedral angle of 24.48 (7)° with the ‘terminal’ benzene ring. Mol­ecules in the crystal are π–π stacked along the [100] direction with a mean inter­planar distance of 3.857 (1) Å. In addition, C—H⋯O inter­actions link them into double layers parallel to the ac plane.
doi:10.1107/S160053681103193X
PMCID: PMC3200752  PMID: 22065404
14.  1-Benzyl­sulfanyl-2-[(2-chloro­phen­yl)diazen­yl]benzene 
The title compound, C19H15ClN2S, a divalent organosulfur compound belonging to the class of ortho-mercaptoazo compounds, is non-ionic in nature. The azo group in the mol­ecule is moved away from the S atom to attain the stable trans-azo configuration. Here the S atom is not electron deficient, so no intra­molecular N⋯S inter­action exists. Due to steric reasons, the mol­ecule is non-planar: the chlorophenyl and benzyl rings are oriented at dihedral angles of 3.21 (8) and 78.18 (5)°, respectively, with respect to the thiophenyl ring. There are no hydrogen bonds and the crystal structure is stabilized by van der Waals inter­actions.
doi:10.1107/S1600536810025730
PMCID: PMC3007467  PMID: 21588269
15.  Reprogramming an ATP-driven protein machine into a light-gated nanocage 
Nature nanotechnology  2013;8(12):10.1038/nnano.2013.242.
Natural protein assemblies have many sophisticated architectures and functions, creating nanoscale storage containers, motors and pumps1–3. Inspired by these systems, protein monomers have been engineered to self-assemble into supramolecular architectures4 including symmetrical5,6, metal-templated7,8 and cage-like structures8–10. The complexity of protein machines, however, has made it difficult to create assemblies with both defined structures and controllable functions. Here we report protein assemblies that have been engineered to function as light-controlled nanocontainers. We show that an adenosine-5′-triphosphate (ATP)-driven group II chaperonin11,12, which resembles a barrel with a builtin lid, can be reprogrammed to open and close on illumination with different frequencies of light. By engineering photoswitchable azobenzene-based molecules into the structure, light-triggered changes in interatomic distances in the azobenzene moiety are able to drive large-scale conformational changes of the protein assembly. The different states of the assembly can be visualized with single particle cryo-electron microscopy, and the nanocages can be used to capture and release non-native cargos. Similar strategies switching atomic distances with light could be used to build other controllable nanoscale machines.
doi:10.1038/nnano.2013.242
PMCID: PMC3859876  PMID: 24270642
16.  1-(4-Hy­droxy­phen­yl)-2-(2-oxidonaphthalen-1-yl)diazen-1-ium methanol hemisolvate 
In the title compound, C16H12N2O2·0.5CH3OH, the H atom of the –OH group has been transfered to the N atom in the azo group, forming a zwitterion. Hence, there is an intra­molecular N—H⋯O, rather than an O—H⋯N, hydrogen bond in the mol­ecule. The mol­ecule is almost planar, the dihedral angle between the benzene ring and the mean plane of the naphthalene ring system being 4.51 (6)°. In the crystal, mol­ecules are linked to and bridged by O—H⋯O hydrogen bonds involving the methanol mol­ecule, which is located about a twofold rotation axis, and hence half-occupied, forming zigzag chains along [001]. Mol­ecules are also linked via C—H⋯π and π–π inter­actions, the latter involving adjacent benzene and naphthalene rings and having a centroid–centroid distance of 3.6616 (13) Å, forming a three-dimensional network.
doi:10.1107/S1600536813012245
PMCID: PMC3685028  PMID: 23795047
17.  Using the Internal Stress Concept to Assess the Importance of Moisture Sorption-induced Swelling on the Moisture Transport through the Glassy HPMC Films 
AAPS PharmSciTech  2008;9(3):891-898.
The purpose of this research was to elucidate the significance of the changes in the mechanical and the volumetric properties on the moisture diffusivity through the polymer films. The internal stress concept was adapted and applied to estimate the relative impact of these property changes on the total stress experienced by a polymer film during storage. Hydroxypropyl Methylcellulose free films were used as a model material prepared at various conditions and stored at different relative humidities. The changes in the internal stress of these films due to the moisture sorption were studied. It was demonstrated that the stress-relaxation of the films increases at increasing moisture content. At the point when there is a definite loss of stress in the film, which is at moisture content higher than 6%, was shown to correlate with the significant increase of the moisture diffusivity. Further investigations revealed that the loss of stress is especially due to the swelling of the polymer rather than the changes in the inherent strain (the quotient between the tensile strength and the modulus of elasticity) of the HPMC films. This implies that the impact of the moisture sorption on the diffusivity is predominantly via volume addition rather than via altering the mechanical properties. Additionally, the approach presented here also brings up a new application of the internal stress concept, which in essence suggests the possibility to estimate the diffusion coefficient from the sorption isotherm and the mechanical analysis data.
doi:10.1208/s12249-008-9128-7
PMCID: PMC2977037  PMID: 18668371
HPMC; internal stress; mechanical properties; moisture diffusion; swelling
18.  4-[(3-Formyl-4-hydroxy­phen­yl)diazen­yl]-N-(pyrimidin-2-yl)benzene­sulfonamide 
The title mol­ecule, C17H13N5O4S, has a trans configuration with respect to the diazenyl (azo) group. The pyrimidine ring and the terminal benzene ring are inclined at angles of 89.38 (4) and 1.6 (6)°, respectively, with respect to the central benzene ring. The conformation of the mol­ecule is in part stabilized by an intra­molecular O—H⋯O hydrogen bond. In the crystal structure, mol­ecules related through inversion centers form hydrogen-bonded dimers involving the sulfon­amide N—H group and the N atom of the pyrimidine ring.
doi:10.1107/S1600536808024239
PMCID: PMC2960714  PMID: 21201664
19.  Characterization of Films with Thickness Less than 10 nm by Sensitivity-Enhanced Atomic Force Acoustic Microscopy 
We present a method for characterizing ultrathin films using sensitivity-enhanced atomic force acoustic microscopy, where a concentrated-mass cantilever having a flat tip was used as a sensitive oscillator. Evaluation was aimed at 6-nm-thick and 10-nm-thick diamond-like carbon (DLC) films deposited, using different methods, on a hard disk for the effective Young's modulus defined as E/(1 - ν2), where E is the Young's modulus, and ν is the Poisson's ratio. The resonant frequency of the cantilever was affected not only by the film's elasticity but also by the substrate even at an indentation depth of about 0.6 nm. The substrate effect was removed by employing a theoretical formula on the indentation of a layered half-space, together with a hard disk without DLC coating. The moduli of the 6-nm-thick and 10-nm-thick DLC films were 392 and 345 GPa, respectively. The error analysis showed the standard deviation less than 5% in the moduli.
doi:10.1007/s11671-010-9778-8
PMCID: PMC3211417
Atomic force acoustic microscopy; Thin film; Elastic modulus; Diamond-like carbon; Concentrated-mass cantilever
20.  Bis{6-[4-(2-ethyoxyphenyl­diazen­yl)phen­oxy]hexa­nol} monohydrate 
The asymmetric unit of the title compound, 2C20H26N2O3·H2O, contains two independent mol­ecules and one water mol­ecule. The azo bonds adopt trans conformations and the dihedral angles between the aromatic rings in the two organic mol­ecules are 4.5 (2) and 1.5 (2)°. In the crystal structure, O—H⋯O and C—H⋯O hydrogen bonds help to establish the packing.
doi:10.1107/S1600536808043924
PMCID: PMC2968466  PMID: 21582172
21.  4-[(E)-1-Naphthyl­diazen­yl]phenol 
The title compound (C. I. Solvent Yellow 8), C16H12N2O, crystallizes with two crystallographically independent mol­ecules in the asymmetric unit. The planarity of both mol­ecules is slightly distorted, the dihedral angles between the benzene ring and the naphthalene system being 9.04 (8) and 5.69 (3)°. In the crystal, O—H⋯N hydrogen bonds between the hydr­oxy groups and azo N atoms link the two symmetry-independent mol­ecules into a polymeric chain propagating in [001].
doi:10.1107/S1600536809009866
PMCID: PMC2968944  PMID: 21582541
22.  Microrheological Characterization of Collagen Systems: From Molecular Solutions to Fibrillar Gels 
PLoS ONE  2013;8(8):e70590.
Collagen is the most abundant protein in the extracellular matrix (ECM), where its structural organization conveys mechanical information to cells. Using optical-tweezers-based microrheology, we investigated mechanical properties both of collagen molecules at a range of concentrations in acidic solution where fibrils cannot form and of gels of collagen fibrils formed at neutral pH, as well as the development of microscale mechanical heterogeneity during the self-assembly process. The frequency scaling of the complex shear modulus even at frequencies of ∼10 kHz was not able to resolve the flexibility of collagen molecules in acidic solution. In these solutions, molecular interactions cause significant transient elasticity, as we observed for 5 mg/ml solutions at frequencies above ∼200 Hz. We found the viscoelasticity of solutions of collagen molecules to be spatially homogeneous, in sharp contrast to the heterogeneity of self-assembled fibrillar collagen systems, whose elasticity varied by more than an order of magnitude and in power-law behavior at different locations within the sample. By probing changes in the complex shear modulus over 100-minute timescales as collagen self-assembled into fibrils, we conclude that microscale heterogeneity appears during early phases of fibrillar growth and continues to develop further during this growth phase. Experiments in which growing fibrils dislodge microspheres from an optical trap suggest that fibril growth is a force-generating process. These data contribute to understanding how heterogeneities develop during self-assembly, which in turn can help synthesis of new materials for cellular engineering.
doi:10.1371/journal.pone.0070590
PMCID: PMC3732230  PMID: 23936454
23.  The mechanical properties of a surface-modified layer on poly(dimethylsiloxane) 
Journal of materials research  2008;23(1):37-48.
Surface-modification of the elastomer poly(dimethylsiloxane) by exposure to oxygen plasma for four minutes creates a thin, stiff film. In this study, the thickness and mechanical properties of this surface-modified layer were determined. Using the phase image capabilities of a tapping-mode atomic-force microscope, the surface-modified region was distinguished from the bulk PDMS; specifically, it suggested a graded surface layer to a depth of about 200 nm. Load-displacement data for elastic indentation using a compliant AFM cantilever was analyzed as a plate bending on an elastic foundation to determine the elastic modulus of the surface (37 MPa). An applied uniaxial strain generated a series of parallel nano-cracks with spacing on the order of a few microns. Numerical analyses of this cracking phenomenon showed that the depth of these cracks was in the range of 300–600 nm and that the surface layer was extremely brittle, with its toughness in the range of 0.1–0.3 J/m2.
doi:10.1557/JMR.2008.0029
PMCID: PMC2749279  PMID: 19779588
24.  (E)-5,5′-(Diazene-1,2-di­yl)diisophthalic acid N,N-dimethyl­formamide disolvate 
The title compound, C16H10N2O8·2C3H7NO, was synthesized by the reductive condensation reaction of 5-nitro­isophthalic acid in the presence of NaOH. The tetra-acid mol­ecule, which has a crystallographically imposed centre of symmetry, adopts an E configuration with respect to the azo group. In the crystal packing, mol­ecules are linked through inter­molecular O—H⋯O and C—H⋯O hydrogen-bonding inter­actions, forming chains propagating in [20].
doi:10.1107/S1600536808032819
PMCID: PMC2959636  PMID: 21581060
25.  Bis(4-meth­oxy­pyridin-3-yl)diazene 
The asymmetric unit of the title compound, C12H12N4O2, consists of one half-mol­ecule, which is located on a center of inversion. The molecule has a step-like shape; the azo group adopting a trans configuration, with the pyridine rings being parallel-displace.
doi:10.1107/S1600536812018326
PMCID: PMC3344664  PMID: 22590426

Results 1-25 (573140)