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BACKGROUND: The gene expression differs in the nuclei of normal and malignant mammalian cells, and transcription is a critical initial step, which defines the difference. The mechanical properties of transcriptionally active chromatin are still poorly understood. Recently we have probed transcriptionally active chromatin of the nuclei subjected to mechanical stress, by Atomic Force Microscopy (AFM) [1]. Nonetheless, a systematic study of the phenomenon is needed. METHODS: Nuclei were deformed and studied by AFM. Non-deformed nuclei were studied by fluorescence confocal microscopy. Their transcriptional activity was studied by RNA electrophoresis. RESULTS: The malignant nuclei under the study were stable to deformation and assembled of 100-300 nm beads-like units, while normal cell nuclei were prone to deformation. The difference in stability to deformation of the nuclei correlated with DNA supercoiling, and transcription-depended units were responsive to supercoils breakage. The inhibitors of the topoisomerases I and II disrupted supercoiling and made the malignant nucleus prone to deformation. Cell nuclei treatment with histone deacetylase inhibitors (HDACIs) preserved the mechanical stability of deformed malignant nuclei and, at the same time, made it possible to observe chromatin decondensation up to 20-60 nm units. The AFM results were supplemented with confocal microscopy and RNA electrophoresis data. CONCLUSIONS: Self-assembly of transcriptionally active chromatin and its decondensation, driven by DNA supercoiling-dependent rigidity, was visualized by AFM in the mechanically deformed nuclei. GENERAL SIGNIFICANCE: We demonstrated that supercoiled DNA defines the transcription mechanics, and hypothesized the nuclear mechanics in vivo should depend on the chromatin architecture.
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Núcleo Celular , Cromatina , Animais , Cromatina/metabolismo , Núcleo Celular/metabolismo , Microscopia de Força Atômica/métodos , RNA/metabolismo , DNA/metabolismo , MamíferosRESUMO
BACKGROUND: Nuclear rigidity is traditionally associated with lamina and densely packed heterochromatin. Actively transcribed DNA is thought to be less densely packed. Currently, approaches for direct measurements of the transcriptionally active chromatin rigidity are quite limited. METHODS: Isolated nuclei were subjected to mechanical stress at 60 g and analyzed by Atomic Force Microscopy (AFM). RESULTS: Nuclei of the normal fibroblast cells were completely flattened under mechanical stress, whereas nuclei of the cancerous HeLa were extremely resistant. In the deformed HeLa nuclei, AFM revealed a highly-branched landscape assembled of ~400 nm closed-packed globules and their structure was changing in response to external influence. Normal and cancerous cells' isolated nuclei were strikingly different by DNA resistance to applied mechanical stress. Paradoxically, more transcriptionally active and less optically dense chromatin of the nuclei of the cancerous cells demonstrated higher physical rigidity. A high concentration of the transcription inhibitor actinomycin D led to complete flattening of HeLa nuclei, that might be related to the relaxation of supercoiled DNA tending to deformation. At a low concentration of actinomycin D, we observed the intermediary formation of stochastically distributed nanoloops and nanofilaments with different shapes but constant width ~ 180 nm. We related this phenomenon with partial DNA relaxation, while non-relaxed DNA still remained rigid. CONCLUSIONS: The resistance to deformation of nuclear chromatin correlates with fundamental biological processes in the cell nucleus, such as transcription, as assessed by AFM. GENERAL SIGNIFICANCE: A new outlook to studying internal nuclei structure is proposed.
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Núcleo Celular , Cromatina , Humanos , Núcleo Celular/genética , Dactinomicina , DNA , Microscopia de Força Atômica , Células HeLaRESUMO
The area-preserving rule for botanical trees by Leonardo da Vinci is discussed in terms of a very specific fractal structure, a logarithmic fractal. We use a method of the numerical Fourier analysis to distinguish the logarithmic fractal properties of the two-dimensional objects and apply it to study the branching system of real trees through its projection on the two-dimensional space, i.e., using their photographs. For different species of trees (birch and oak) we observe the Q^{-2} decay of the spectral intensity characterizing the branching structure that is associated with the logarithmic fractal structure in two-dimensional space. The experiments dealing with the side view of the tree should complement the area preserving Leonardo's rule with one applying to the product of diameter d and length l of the k branches: d_{i}l_{i}=kd_{i+1}l_{i+1}. If both rules are valid, then the branch's length of the next generation is sqrt[k] times shorter than previous one: l_{i}=sqrt[k]l_{i+1}. Moreover, the volume (mass) of all branches of the next generation is a factor of d_{i}/d_{i+1} smaller than previous one. We conclude that a tree as a three-dimensional object is not a logarithmic fractal, although its projection onto a two-dimensional plane is. Consequently, the life of a tree flows according to the laws of conservation of area in two-dimensional space, as if the tree were a two-dimensional object.
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The small-angle neutron scattering (SANS) on HeLa nuclei demonstrates the bifractal nature of the chromatin structural organization. The border line between two fractal structures is detected as a crossover point at Q_{c}≈4×10^{-2}nm^{-1} in the momentum transfer dependence Q^{-D}. The use of contrast variation (D_{2}O-H_{2}O) in SANS measurements reveals clear similarity in the large scale structural organizations of nucleic acids (NA) and proteins. Both NA and protein structures have a mass fractal arrangement with the fractal dimension of D≈2.5 at scales smaller than 150 nm down to 20 nm. Both NA and proteins show a logarithmic fractal behavior with D≈3 at scales larger than 150 nm up to 6000 nm. The combined analysis of the SANS and atomic force microscopy data allows one to conclude that chromatin and its constitutes (DNA and proteins) are characterized as soft, densely packed, logarithmic fractals on the large scale and as rigid, loosely packed, mass fractals on the smaller scale. The comparison of the partial cross sections from NA and proteins with one from chromatin as a whole demonstrates spatial correlation of two chromatin's components in the range up to 900 nm. Thus chromatin in HeLa nuclei is built as the unified structure of the NA and proteins entwined through each other. Correlation between two components is lost upon scale increases toward 6000 nm. The structural features at the large scale, probably, provide nuclei with the flexibility and chromatin-free space to build supercorrelations on the distance of 10^{3} nm resembling cycle cell activity, such as an appearance of nucleoli and a DNA replication.
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The small-angle neutron scattering (SANS) on the rat lymphocyte nuclei demonstrates the bifractal nature of the chromatin structural organization. The scattering intensity from rat lymphocyte nuclei is described by power law Q^{-D} with fractal dimension approximately 2.3 on smaller scales and 3 on larger scales. The crossover between two fractal structures is detected at momentum transfer near 10^{-1}nm^{-1}. The use of contrast variation (D_{2}O-H_{2}O) in SANS measurements reveals clear similarity in the structural organizations of nucleic acids (NA) and proteins. Both chromatin components show bifractal behavior with logarithmic fractal structure on the large scale and volume fractal with slightly smaller than 2.5 structure on the small scale. Scattering intensities from chromatin, protein component, and NA component demonstrate an extremely extensive range of logarithmic fractal behavior (from 10^{-3} to approximately 10^{-1}nm^{-1}). We compare the fractal arrangement of rat lymphocyte nuclei with that of chicken erythrocytes and the immortal HeLa cell line. We conclude that the bifractal nature of the chromatin arrangement is inherent in the nuclei of all these cells. The details of the fractal arrangement-its range and correlation/interaction between nuclear acids and proteins are specific for different cells and is related to their functionality.
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The small-angle neutron scattering (SANS) on the chicken erythrocyte nuclei demonstrates the bifractal nature of the chromatin structural organization. Use of the contrast variation (D_{2}O-H_{2}O) in SANS measurements reveals the differences in the DNA and protein arrangements inside the chromatin substance. It is the DNA that serves as a framework that constitutes the bifractal behavior showing the mass fractal properties with D=2.22 at a smaller scale and the logarithmic fractal behavior with D≈3 at a larger scale. The protein spatial organization shows the mass fractal properties with D≈2.34 throughout the whole nucleus. The borderline between two fractal levels can be significantly shifted toward smaller scales by centrifugation of the nuclei disposed on the dry substrate, since nuclei suffer from mechanical stress transforming them to a disklike shape. The height of this disk measured by atomic force microscopy (AFM) coincides closely with the fractal borderline, thus characterizing two types of the chromatin with the soft (at larger scale) and rigid (at smaller scale) properties. The combined SANS and AFM measurements demonstrate the stress induced switch of the DNA fractal properties from the rigid, but loosely packed, mass fractal to the soft, but densely packed, logarithmic fractal.
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Núcleo Celular/genética , DNA/metabolismo , Eritrócitos/citologia , Fractais , Estresse Mecânico , Animais , Fenômenos Biomecânicos , Galinhas , Microscopia de Força Atômica , Modelos BiológicosRESUMO
The phonon dispersion of FeSi was measured by inelastic x-ray scattering. The study of its temperature evolution in the range of 100 K-300 K showed that the phonon modes soften to a different extent. The phonons exhibiting specifically strong softening were revealed to belong to the weakly dispersive optical branch. At the same time, the calculations of the lattice dynamics of FeSi suggest that this branch corresponds mainly to the atomic displacements that change the Fe-Fe nearest neighbor distance. It points to the role of the Fe-Fe interaction in the phonon softening.
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Single crystal synchrotron diffraction for pressures up to 50 GPa has revealed an essential difference in structural properties and compressibility of MnGe compared with Mn1-x Co x Ge and Mn1-x Fe x Ge solid solutions. A negative thermal expansion has been observed for MnGe at low-temperatures and high-pressures. The single crystal refinement has shown a discontinuous change of the atomic coordinates and Mn-Ge interatomic distances of MnGe in contrast to Mn0.1Co0.9Ge. These peculiarities of MnGe are likely to be associated with high-spin-low-spin transition. The relation between anisotropy of the coordination of Mn-atom and its magnetic moment is discussed.
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Small-angle neutron scattering (SANS) on nuclei of chicken erythrocytes demonstrates the cubic dependence of the scattering intensity Q^{-3} in the range of momentum transfer Q∈10^{-3}-10^{-2}nm^{-1}. Independent spin-echo SANS measurements give the spin-echo function, which is well described by the exponential law in a range of sizes (3×10^{2})-(3×10^{4}) nm. Both experimental dependences reflect the nature of the structural organization of chromatin in the nucleus of a living cell, which corresponds to the correlation function γ(r)=ln(ξ/r) for r<ξ, where ξ=(3.69±0.07)×10^{3} nm, the size of the nucleus. It has the specific scaling property of the logarithmic fractal γ(r/a)=γ(r)+ln(a), i.e., the scaling down by a gives an additive constant to the correlation function, which distinguishes it from the mass fractal, which is characterized by multiplicative constant.
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Núcleo Celular/química , Cromatina/química , Eritrócitos/química , Modelos Biológicos , Animais , Núcleo Celular/metabolismo , Galinhas , Cromatina/metabolismo , DNA/química , DNA/metabolismo , Eritrócitos/metabolismo , Fractais , Modelos Moleculares , Difração de Nêutrons , Conformação de Ácido Nucleico , Espalhamento a Baixo ÂnguloRESUMO
Temperature dependent powder and single-crystal synchrotron diffraction, specific heat, magnetic susceptibility and small-angle neutron scattering experiments have revealed an anomalous response of MnGe. The anomaly becomes smeared out with decreasing Mn content in Mn1-x Co x Ge and Mn1-x Fe x Ge solid solutions. Mn spin state instability is discussed as a possible candidate for the observed effects.
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Separating between the ordinary Hall effect and anomalous Hall effect in the paramagnetic phase of Mn_{1-x}Fe_{x}Si reveals an ordinary Hall effect sign inversion associated with the hidden quantum critical (QC) point x^{*}â¼0.11. The effective hole doping at intermediate Fe content leads to verifiable predictions in the field of fermiology, magnetic interactions, and QC phenomena in Mn_{1-x}Fe_{x}Si. The change of electron and hole concentrations is considered as a "driving force" for tuning the QC regime in Mn_{1-x}Fe_{x}Si via modifying the Ruderman-Kittel-Kasuya-Yosida exchange interaction within the Heisenberg model of magnetism.
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Magnetic susceptibility measurements have shown that the compounds Mn(1-x)Fe(x)Ge are magnetically ordered through the whole range of concentrations x = [0.0,1.0]. Small-angle neutron scattering reveals the helical nature of the spin structure with a wave vector, which changes from its maximum (|k| = 2.3 nm(-1)) for pure MnGe, through its minimum (|k| â 0) at x(c) ≈ 0.75, to the value of |k| = 0.09 nm(-1) for pure FeGe. The macroscopic magnetic measurements confirm the ferromagnetic nature of the compound with x = x(c). The observed transformation of the helix structure to the ferromagnet at x = x(c) is explained by different signs of chirality for the compounds with x > x(c) and x
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We show, with the help of polarized neutrons, that the cubic magnets Fe1-xCoxSi with Dzyaloshinskii-Moriya interaction can be switched between left (for x=0.1, 0.15) and right (for x=0.2, 0.25, 0.3, 0.5) chiral states of the spin helix. The absolute structure was evaluated using x-ray diffraction. The crystals are shown to be enantiopure and the structural chirality changes from right handed for x<0.2 to left handed for x>0.2. These compounds are compared with the etalon sample of MnSi which is identified as having the left-handed chirality both in the magnetic and crystallographic sense.
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Polarized neutron scattering experiments have demonstrated that Dy/Y multilayer structures possess a coherent spin helix with a preferable chirality induced by the magnetic field. The average chirality, being proportional to the difference in the left- and right-handed helix population numbers, is measured as a polarization-dependent asymmetric part of the magnetic neutron scattering. The magnetic field applied in the plane of the sample upon cooling below T(N) is able to repopulate the otherwise equal population numbers for the left- and right-handed helixes. The experimental results strongly indicate that the chirality is caused by Dzyaloshinskii-Moriya interaction due to the lack of the symmetry inversion on the interfaces.