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Experimental and computational efforts are constantly made to elucidate mechanisms controlling cell fate decisions during development and cell reprogramming. One powerful method is to consider cell commitment and reprogramming as movements in an energy landscape. Here, we develop CELLoGeNe (Computation of Energy Landscapes of Logical Gene Networks), which maps Boolean implementation of gene regulatory networks (GRNs) into energy landscapes. CELLoGeNe removes inadvertent symmetries in the energy landscapes normally arising from standard Boolean operators. Furthermore, CELLoGeNe provides a tool for visualising multi-dimensional energy landscapes and a platform to stochastically probe and analyse the shapes of the computed landscapes corresponding to the epigenetic landscapes for development and reprogramming. We demonstrate CELLoGeNe on a GRN governing maintenance and self-renewal of pluripotency, identifying attractors experimentally validated. We also apply CELLoGeNe on a network controlling reprogramming from mouse embryonic fibroblast (MEF) to induced pluripotent stem cells (iPSCs) where we identify potential roadblocks as attractors. CELLoGeNe is a general framework that can be applied to various biological systems offering a broad picture of intracellular dynamics otherwise inaccessible with existing methods.

The generation of induced pluripotent stem cells (iPSCs) with Oct4, Sox2, Klf4, c-Myc has been described as 'direct' reprogramming in contrast to reprogramming via nuclear transfer. Interestingly, recent studies have suggested that the conversion process itself includes transient up-regulation and down-regulation of hundreds of genes, making unique intermediate populations. In a sense, the process of 4 factor reprogramming is indirect. Like in vitro differentiation, iPSC generation efficiency and kinetics largely depend on the external environment, as well as the amount and stoichiometry of exogenously expressed reprogramming factors. However, accumulating evidence indicates that when reprogramming succeeds, the process is not random but progresses in an ordered, step-wise manner. In this review, we summarize current knowledge detailing how somatic cells reach a pluripotent state.

One of the most popular crystalline morphologies is a spherulite. An evidence is reported that the spherulite is crystallized through a dense packing state of small particles appearing in a droplet, which is caused by the primary phase separation of the melt in the metastable region of a phase diagram proposed by Olmsted et al. [Olmsted PD, Poon WCK, McLeish TCB, Terrill NJ, Ryan AJ. Phys Rev Lett 1998; 81: 373]. According to this phase diagram, the crystallization from the metastable state causes the nucleation and growth (N & G) of nematic domains, here named droplets, in the isotropic matrix. As a next step, the secondary phase separation of spinodal decomposition (SD) type into smectic and amorphous domains occurs inside the droplet where entanglements are excluded from the smectic to the amorphous domain; then such an SD structure turns into a densely packing structure of many small particles owing to surface tension. At this final stage of the induction period a long period peak of small-angle X-ray scattering (SAXS), so-called SAXS before WAXS, appears, which may be due to the average distance between these small particles. Furthermore, it is considered that crystalline lamellae are formed by radial and azimuthal fusion of these small particles inside the droplet, resulting in a spherulite. Such a type of crystallization occurs most commonly when flexible polymers are crystallized under the usual conditions. This tentative concept of spherulitic growth, which is completely different from a theory by Keith and Padden [Keith HD, Padden FJ. J Appl Phys 1963; 34: 2409], would give a new insight into problems of spherulites.

Novel morphology of isotactic polypropylene (iPP) crystal, which is quite different from that of usual spherulite, has been observed by scanning electron microscope (SEM). The crystals show ‘bamboo leaf-like (BL)’ shape with α-monoclinic high crystallinity. The BL crystals are formed by neither melt nor glass crystallization, but by a complicated annealing process that goes through mesomorphic phase of iPP. Substrates are not essential for the formation of BL crystals, since the BL crystals are formed both on glass surface and free surface as well as in bulk. Along with the annealing process, a possible explanation for the mechanism of the formation of the BL crystal is proposed.

CD9 is a membrane protein belonging to the tetraspanin family. Despite CD9's broad tissue distribution, the only abnormality observed in CD9-deficient mice was infertility of females, which was responsible for a defect in the sperm-egg fusion process. However, the function of CD9 in sperm-egg fusion is not clear at all because the technique to analyze the activity of molecules in sperm-egg fusion has not been established. We demonstrated that the exogenous mouse CD9, expressed by polyadenylated mRNA injection at the germinal-vesicle stage oocytes, was precisely localized to the egg plasma membrane, and the expression reversed the infertility of CD9(-/-) eggs. Then, two other tetraspanins, human CD9 and mouse CD81, overexpressed with this technique on CD9(-/-) eggs restored the fertilization rate up to approximately 90 and approximately 50% against that of wild type eggs, respectively. Moreover, in the presence of an anti-mouse CD9 mAb, which blocks sperm-egg fusion, expression of human CD9 or mouse CD81 on eggs also rescued the fusibility. These results suggested that human CD9 plays a crucial role in human fertilization, and mouse CD81 has the potential to compensate for CD9 function in sperm-egg fusion. In addition, the polyadenylated mRNA injection is effective for molecular analysis of sperm-egg fusion.

Intramolecular hydrodynamic contribution ηintra/C to the reduced viscosity ηSP/C of polyelectrolyte solutions is derived as a function of polymer concentration C by separating the theoretically calculated intermolecular electrostatic contribution ηinter/C from the observed reduced viscosity, assuming an additivity, ηSP/C=ηintra/C+ηinter/C. The resulting intramolecular part ηintra/C reflects nearly the net effect of the polyion conformation; it increases monotonously with decreasing polymer concentration and levels off to a constant in sufficiently dilute concentrations. The leveling-off value of ηintra/C corresponds to the intrinsic viscosity [η]. From the estimated values of [η], the ionic strength I dependence of the polyion conformation has been visualized, resulting in a similarity between two relations, ηintra/C vs. C and [η] vs. I.

Intermolecular electrostatic interactions of polyions contributing to the reduced viscosity are calculated for two molecular models of charged points and skewed rods. These simplified models approximately describe inter-polyion configurations in the actual polyelectrolyte solutions to qualitatively reproduce the characteristic peak of the reduced viscosity appearing at lower concentrations. The long-standing theoretical equation by Rice and Kirkwood was adopted for this calculation because of its simplicity. For the potential term of this equation, we assumed a Debye type screening potential as a function of ionic strength. The results show that the skewed rods model well fits with the experimental data. Furthermore, it should be pointed out that the configurations of this model correspond to the basic constituents of the so-called isotropic model proposed by de Gennes et al.

The compatibility of PSSNa and PVSNa in aqueous solution has been studied. PSSNa and PVSNa are compatible when the total concentration is less than 1.0 mol/l, but phase separation occurs when the total concentration exceeds 1.0 mol/l. The cloud point curve is of a typical UCST type. Addition of salt causes a shift of the boundary toward a lower concentration. It may be considered that these new critical phenomena are a reflection of the balance between electrostatic and non-electrostatic interactions. The time evolving processes of the phase separation has also been investigated. The characteristic behavior of spinodal decomposition is observed when the temperature of a mixed solution is jumped down passing through the critical point.

Osteoclast progenitors differentiate into mature osteoclasts in the presence of receptor activator of NF-kappaB (RANK) ligand on stromal or osteoblastic cells and monocyte macrophage colony-stimulating factor (M-CSF). The soluble RANK ligand induces the same differentiation in vitro without stromal cells. Tumor necrosis factor-alpha (TNF-alpha), a potent cytokine involved in the regulation of osteoclast activity, promotes bone resorption via a primary effect on osteoblasts; however, it remains unclear whether TNF-alpha can also directly induce the differentiation of osteoclast progenitors into mature osteoclasts. This study revealed that TNF-alpha directly induced the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (MNCs), which produced resorption pits on bone in vitro in the presence of M-CSF. The bone resorption activity of TNF-alpha-induced MNCs was lower than that of soluble RANK ligand-induced MNCs; however, interleukin-1beta stimulated this activity of TNF-alpha-induced MNCs without an increase in the number of MNCs. In this case, interleukin-1beta did not induce TRAP-positive MNC formation. The osteoclast progenitors expressed TNF receptors, p55 and p75; and the induction of TRAP-positive MNCs by TNF-alpha was inhibited completely by an anti-p55 antibody and partially by an anti-p75 antibody. Our findings presented here are the first to indicate that TNF-alpha is a crucial differentiation factor for osteoclasts. Our results suggest that TNF-alpha and M-CSF play an important role in local osteolysis in chronic inflammatory diseases.

Local mobility of three substituted polyacetylenes was investigated by means of a quasielastic neutron scattering technique aiming at elucidating the origin of gas permeability of these polymers. It was found that the local flux, which is defined as the product of the relaxation rate Γ, and the mobile fraction f m , increases with the gas permeability coefficient. This suggests that the local flux is an important factor controlling gas permeability.

A novel crystal morphology called terrace appearing in the crystallization of poly(ethylene terephthalate) (PET) is studied. This morphology is confirmed to be inherent to the polymer itself and is formed when the polymer crystallizes from a very thin melt film. The growing processes of the terrace are examined in detail using confocal scanning laser microscopy (CSLM) and atomic force microscopy (AFM). The terraces appear on the periphery of a two-dimensional spherulite at the final stage of the spherulite growth. They have a single crystal-like structure though their growth front or outer shape is not smooth but fluctuating; it shows a periodic pattern characteristic of a diffusion-controlled process. AFM reveals that the terrace is about 13 nm thick and its growth front is in contact with a further thinner melt layer about 2–4 nm thick, suggesting that amorphous polymer molecules are supplied to the growing front only from the thinner melt layer. As crystallization proceeds, long needle-like crystals protrudes from the terrace front, resulting in the so-called finger pattern. Such needle growth is then suppressed by accompanying the tip-splitting of some needle crystals to produce crabs pincers-like crystals. Monte Carlo simulations for the two-dimensional diffusion model reproduce the morphologies, where the surface tension and the crystal anisotropy are shown to be crucially important.

On the basis of the neutron data and the heat capacities, a characteristic length, ξ, or spatial scale of the low-energy excitation mode has been evaluated for poly(methyl methacrylate) (PMMA), polystyrene (PS) and polyethylene (PE) in the amorphous state in terms of the soft potential model. We discuss a possible structural origin of the low-energy excitation in connection with the cooperatively rearranging region in the supercooled state.

We studied the structure of poly(vinyl alcohol) (PVA) gels formed in mixtures of dimethyl sulfoxide (DMSO) and water using several scattering techniques such as wide-angle neutron scattering (WANS), small-angle neutron scattering (SANS), ultra-small-angle neutron scattering (U-SANS) and light scattering (LS) to cover a very wide Q range from 10 -4 to 10 A -1 . The WANS measurements have revealed that the cross-linking points of the gels are crystallites, and the size and its distribution have been evaluated by the SANS measurements. The SANS results have also shown that the structure observed in the low Q range below 10 -2 A -1 is dominated by a liquid–liquid-phase separation. The early stage of the phase separation has been studied in detail using the time-resolved LS technique, while the late stage has been investigated by the U-SANS technique because the LS measurements cannot access the opaque samples. On the basis of the results, we present a quantitative sketch of the structure of the PVA gel.

Low-energy excitations of amorphous and semicrystalline poly(ethylene terephthalate)s (PETs) have been studied by inelastic and quasielastic neutron scattering in an energy range of 0.2–10 meV. It was found that the low-energy excitation is not affected by the density fluctuations observed around Q = 0.03 A −1 in the amorphous state. It was also found that dynamic scattering law Sc(Q, ω) of the crystalline phase is not described by the Debye theory, suggesting that the crystalline phase includes many disordered regions from which the low-energy excitation is originated.

The dynamics of amorphous polystyrene (PS) near the glass transition temperature Tg( = 373 K) have been studied by quasielastic neutron scattering in the energy range from 0.1 to 10 meV. In order to elucidate the role of the side-chain motion for the glass-transition dynamics, we observed the motion of the side chains (phenyl groups) separately from that of the main chain by measuring PS with deuterated side chains (PS-d5), in addition to hydrogenated PS. The observed dynamic scattering laws were analyzed by a curve-fitting method, assuming a phenomenological model function consisting of three components: a δ-function, a Lorentzian and a flat background. It was found that the widths of the Lorentzian are identical for PS and PS-d5 near Tg, suggesting strong dynamical coupling between the side and main chains.

Dynamics of amorphous polymers have been studied below and above the glass transition temperature, T g , by inelastic and quasielastic neutron scattering technique in the energy range of 0.02 to 10 meV. All the amorphous polymers examined show a broad excitation peak, the so-called ‘low energy excitation’ peak, at 1.5–4 meV below T g , irrespective of differences in chemical structures. As temperature increases, neutron spectra become quasielastic-like (spectral softening) due to onset of a very fast motion of ps order. The fast motion appears at about 50 K below T g for cis-1,4-polybutadiene, which is close to the Vogel-Fulcher temperature, T 0 . On the other hand, atactic polystyrene (a-PS) shows the spectral softening at about 200 K, being about 170 K below T g . The origin of the spectral softening of a-PS was assigned to onset of side chain motion.

We constructed a crystal analyzer type inelastic scattering neutron spectrometer LAM-D at the KENS spallation thermal neutron source in the National Laboratory for High Energy Physics (KEK), Tsukuba, Japan. This spectrometer can cover the energy transfer range up to 300 meV with a resolution ΔϵE1 of less than 6%. The basic principle of LAM-D is the same as the quasi-elastic scattering spectrometers LAM-40 and LAM-80 at KENS so that the combination of the three spectrometers is a very powerful tool for studies of molecular motions in condensed matters in the energy range of 1 μeV to 300 meV.

Local dynamics of cis-1, 4-polybutadiene near the glass transition temperature Tg (=170K) has been investigated by quasielastic neutron scattering in the energy range of 0.01 meV to 10meV. It was found that two models of motions appear near the Tg in the energy range of ca. 1 meV and 10 ∽ 30 μeV. The fast mode arises ca. 50 K below Tg which corresponds to the so-called Vogel temperature T0. On the other hand, the slow mode appears just above Tg. The natures of the two modes are discussed from viewpoints of glass transition.

A new finding is reported concerning some structural change in the amorphous state of the polymer which occurs during the so-called induction period before the start of crystallization. Annealing of poly(ethylene terephthalate) (PET) at 115°C, or 40°C above the glass transition temperature, Tg, has been investigated by small-angle X-ray scattering (SAXS) and wide-angle X-ray scattering (WAXS) techniques. At a very early stage of the induction period the SAXS intensity starts to increase in the range of Q = 0.02–0.04 A −1 , where Q is the length of the scattering vector (Q = 4π sin θ/λ), and continues to increase until the beginning of crystallization. Corresponding to the initation of crystallization, another strong peak, caused by the usual long-period structure, appears in the vicinity of Q = 0.05 A −1 . It is confirmed from the WAXS measurements that no local ordering takes place during the induction period. To clarify the crystallization process, distance distribution analysis is also carried out.

Apparent gelation rates t−1gel, defined as the reciprocal of the gelation time, are measured for poly(vinyl alcohol) solutions as functions of polymer concentration, Cp, and quenching temperature, TQ. Here the measurements are limited to the case of homogeneous gelation where TQ is below 0°C. The critical gelation concentration, C∗ t = ∞ , extrapolated to t−1gel = 0 is independent of TQ, and is identical to the polymer chain overlap concentration, C∗ Rg . In order to represent the degree of polymer chain overlap a reduced concentration, o = (C p - C∗ Rg ) C∗ Rg , is introduced. An experimental relation, t−1gel = A(TQ) o2, is obtained where A(TQ) is the temperature factor. The o dependence of the equation can be explained using the chain overlap concept for gelation and random bond percolation theory, and the temperature factor A(TQ) is analysed by the crystal nucleation theory.

Crystallization processes of poly(ethylene terephthalate) (PET) when annealed at 80°C, or 5°C above the glass transition temperature, Tg, have been investigated by a small-angle X-ray scattering (SAXS) technique. At a very early stage of annealing, a scattering maximum appears at around Q = 0.04 A −1 , where Q is the length of the scattering vector (Q = 4π sin θ/λ). During the so-called induction period of crystallization, this maximum intensity increases with annealing time and the maximum position shifts towards the low Q side. These results confirm the previously reported new finding that the long-range ordered structure is formed in the induction period before crystallization. These ordering processes can be divided into two stages: an early and a late stage. The scattering behaviour in the early stage is in accordance with the prediction of Cahn's linearized theory for spinodal decomposition. The scattering profiles in the late stage can be described in terms of Furukawa's scaling theory for the cluster growth regime. From these experimental results, we conclude that the growth process of the density fluctuation occurs in the induction period and this process is very similar to the spinodal decomposition type of phase separation process. It is considered that after this dense domain grows to a certain size, crystallization begins.

We report the quasielastic neutron scattering results on an amorphous polyimide below the glass transition temperature Tg (=523 K). The so-called fast process in picosecond order has been observed far below Tg. We also performed dynamic-mechanical measurements at 10 Hz to find that the β-relaxation temperature is close to the onset temperature of the fast process although the difference in the frequencies in both the measurements are more than ∼1010 Hz.

Neutron spin echo measurements were performed on polymeric micelles consisting of deuterated polystyrene (d-PS)-polyisoprene (PI) diblock copolymers in dilute and concentrated solutions. The scattering length density of the core of the micelles matched to the solvent so that dynamics of polymer chains in the corona were measured. The observed intermediate scattering functions in the dilute solution were analyzed by the theory for tethered chains proposed by de Gennes. In the concentrated solutions, motional slowing down was observed, which may be caused not only by increase of the viscosity but also by decrease of the osmotic compressibility.

Low energy excitations in crystalline and amorphous solids of Tri- O -methyl-β-cyclodextrin (TMCD) were studied by neutron scattering and calorimetric techniques. Amorphous solids of TMCD were prepared by two different ways: mechanical milling and liquid-quenching amorphization. Both the amorphous solids showed a boson peak at around 2 meV and there was no significant difference between them. It was noted that crystalline TMCD also showed a boson peak. This anomalous boson peak in the crystalline state was confirmed by low temperature heat capacity measurements.

The electrostatic persistence lengths bc of NaPSS polyions have been measured as a function of ionic strength I in the solutions using a small-angle neuron scattering (SANS) technique combined with a zero average contrast (ZAC) method. This ZAC method provides the optical theta condition distinguishing the intermolecular scattering functions S2(q), and thereby the scattering functions of a single chain S1(q) free from the intermolecular interferences were obtained. The resulting bc values which were derived by the analysis of S1(q) are proportional to I−12. This work confirms and extends an earlier SANS study where the contrast-match was used.

A converging collimator with NiC/Ti supermirrors has been mounted on the high-resolution neutron spectrometer LAM-80ET (Δ ɛ ∼ 1.2 μeV) installed at the pulsed cold-neutron source in the National Laboratory for High Energy Physics (KEK), Tsukuba, Japan. The design and the performance of the collimator are described.

The change of chain conformation during the induction period of crystallization of poly(ethylene terephthalate) (PET) has been studied by means of a deuterium-labelling technique with small-angle neutron scattering. The scattering function of a single chain was analyzed based on the worm-like chain model. The stiffness of PET chains increased in the early stage of the induction period. According to the isotropic-nematic transition theory proposed by Doi et al., this increase of the chain stiffness induces the parallel ordering of polymer segments due to the excluded-volume effect. This orientational ordering may play an important role in the growth process of the density fluctuations observed in the induction period of crystallization.

The local dynamics of amorphous polystyrene have been investigated by incoherent inelastic and quasielastic neutron scattering in a temperature range of 20 to 300 K, below the glass transition temperature T g ( = 373 K). The elasticscattering intensity shows an anomalous decrease at temperatures above 200 K. Corresponding to the anomalous decrease, the shape of the dynamic scattering law S ( Q,ω ) changes from inelastic-like to quasielastic-like, suggesting the onset of a fast relaxation process. On the basis of the results from hydrogenated polystyrene and polystyrene with a deuterated phenyl ring, the fast relaxation process occurring far below T g has been assigned to motion of the phenyl side chains.

The sol-gel-glass transition of atactic polystyrene in carbon disulfide over a wide temperature range from 170 to 300 K has been investigated by neutron quasielastic scattering methods. A drastic narrowing of the quasielastic peaks was observed for the present system as the temperature is lowered and the narrowing completes at the glass transition. Two types of local motion were observed in the sol-gel transition.

Low energy excitations in amorphous polystyrene have been studied at 10 K using inelastic neutron scattering in the ω range of 0.2–10 meV.

Structural studies have been performed on poly(vinyl alcohol)(PVA) gels formed in mixtures of dimethylsulfoxide and water at 23°C by means of small angle and wide angle neutron scattering techniques in a wide Q range of 0.003–10A-1.

The structure of the nucleosome core particle at low ionic strenth (10-0.04 mM Na + ) has been studied by small angle neutron scattering in various H 2 O/D 2 O mixtures. At 0.04 mM Na + ,the radius of gyration obtained at 39% D 2 O where scattering from the DNA dominates was 32 A, and at 65% D 2 O where scattering from the histones dominates, 40 A. An abrupt increase in the radius of gyration from 35 to 40 A was observed at about 1 mM ionic strength at 65% D 2 O, with decreasing the ionic strength from 10 mM. No loss of the histone secondary structure was detected by circular dichroism over the range of the ionic strength examined. These results suggest that at low ionic strength (⪅1 mM) the histones are located outside of the nucleosome core particle accompanied by an alteration of the tertiary and/or quaternary structure of the histone octamer, with the DNA in a folded conformation.

Neutron inelastic scattering measurements were carried out on semicrystalline and highly crystalline polyethylenes with degrees of crystallinity of 0.46 and 0.96, respectively, at 10 K to investigate low-energy excitations below 10 meV. It was found that the density of states G (ω) of the crystalline phase can be described by the Debye model but that of the amorphous phase shows an excess excitation peak at ≈2.5 meV. The calculated heat capacity C ( T ) of the amorphous phase shows a peak at ≈ 6 K in the plot of C ( T )/ T 3 versus T , while C ( T )/ T 3 for the crystalline phase is constant at low temperatures.

In order to evaluate the conformation of a single chain in mixed solutions of sodium salts of hydrogenated and deuterated poly(vinyl sulfuric acid) (PVSNa(H) and PVSNa(D)) without added salts, measurements of time-of-flight neutron small angle scattering (NSAS) were performed while making use of a wide band chopper. By means of the deuterium labeling, the scattering law, S(Q) , of the polymer solution can be separated into two parts: the intrapolymer scattering law, S 1 ( Q ), and the interpolymer one, S 2 ( Q ). Using the Kratky plot, it is concluded that at Q values larger than 0.04 A −1 , the polyelectrolyte can be regarded as a rod-like molecule. It has also been shown that the wide band chopper could be useful for NSAS studies of polymer solutions as well as crystalline aggregates of metals.

Two crystal analyser type quasielastic neutron spectrometers, a conventional resolution device with high efficiency, and a high resolution one with a wide energy window, have been installed at a spallation pulsed cold neutron source (a 20 K methane moderator). A data reduction program using a technique of statistical data analysis has also been developed. These spectrometers combined with the data reduction program have demonstrated their usefulness in profile analysis studies for low energy diffusive phenomena in molecular liquids, polymers and other materials.

We have developed a neutron quasielastic scattering spectrometer with conventional energy resolution. The spectrometer operates at the spallation cold neutron source in the KENS spallation source at the National Laboratory for High Energy Physics. High efficiency was achieved by adopting large analyser mirrors and the spallation cold nuetron source. The spectrometer has demonstrated considerable usefulness in spectral profile analysis studies of molecular liquids and polymers.