Early stages of clathrin aggregation at a membrane in coarse-grained simulations.

The self-assembly process of clathrin coated pits during endocytosis has been simulated by combining and extending coarse grained models of the clathrin triskelion, the adaptor protein AP2, and a flexible network membrane. The AP2's core, upon binding to membrane and cargo, releases a motif that can bind clathrin. In conditions where the core-membrane-cargo binding is weak, the binding of this motif to clathrin can result in a stable complex. We characterize the conditions and mechanisms resulting in the formation of clathrin lattices that curve the membrane, i.e., clathrin coated pits. The mechanical properties of the AP2 ß linker appear crucial to the orientation of the curved clathrin lattice relative to the membrane, with wild-type short linkers giving rise to the inward curving buds enabling endocytosis while long linkers produce upside-down cages and outward curving bulges.

Coarse grain forces in star polymer melts.

An analysis is presented of forces acting on the centers of mass of three-armed star polymers in the molten state. The arms consist of 35 Kremer-Grest beads, which is slightly larger than needed for one entanglement mass. For a given configuration of the centers of mass, instantaneous forces fluctuate wildly around averages which are two orders of magnitude smaller than their root mean square deviations. Average forces are well described by an implicit many-body potential, while pair models fail completely. The fluctuating forces are modelled by means of dynamical variables quantifying the degree of mixing of the various polymer pairs. All functions and parameters in a coarse grain model based on these concepts are obtained from the underlying small scale simulation. The coarse model reproduces both the diffusion coefficient and the shear relaxation modulus. Ways to improve the model suggest themselves on the basis of our findings.

Mesoscale modeling of shear-thinning polymer solutions.

We simulate the linear and nonlinear rheology of two different viscoelastic polymer solutions, a polyisobutylene solution in pristane and an aqueous solution of hydroxypropylcellulose, using a highly coarse-grained approach known as Responsive Particle Dynamics (RaPiD) model. In RaPiD, each polymer has originally been depicted as a spherical particle with the effects of the eliminated degrees of freedom accounted for by an appropriate free energy and transient pairwise forces. Motivated by the inability of this spherical particle representation to entirely capture the nonlinear rheology of both fluids, we extended the RaPiD model by introducing a deformable particle capable of elongation. A Finite-Extensible Non-Linear Elastic potential provides a free energy penalty for particle elongation. Upon disentangling, this deformability allows more time for particles to re-entangle with neighbouring particles. We show this process to be integral towards recovering the experimental nonlinear rheology, obtaining excellent agreement. We show that the nonlinear rheology is crucially dependent upon the maximum elongation and less so on the elasticity of the particles. In addition, the description of the linear rheology has been retained in the process.

Inducement by directional fields of rotational and translational phase ordering in polymer liquid-crystals.

The effects of aligning fields on models of polymer liquid crystals were simulated using the dissipative particle dynamics method. Exposing a liquid crystal of rod-like particles to a directional field causes a stabilization of the phases with orientational order, shifts the isotropic-nematic and nematic-smectic-A phase transitions to higher temperatures, makes the transitions continuous beyond a critical field strength, and induces weak para-nematic alignment in the zero-field isotropic phase. The interplay of liquid-crystalline ordering, microphase separation, and an alignment field endows the diblock and triblock copolymers studied here with rich phase behavior. The simulations suggest that field-induced orientational ordering can give rise to positional ordering. Reversely, positional ordering resulting from rod-coil demixing may be accompanied by orientational ordering, which is enhanced by external fields. For highly asymmetric rod-coil copolymers, the microphase separation pattern formed by the rigid segments can be altered by an aligning field.

Coarse-grained simulations of moderately entangled star polyethylene melts.

In this paper, a previous coarse-grain model [J. T. Padding and W. J. Briels, J. Chem. Phys. 117, 925 (2002)] to simulate melts of linear polymers has been adapted to simulate polymers with more complex hierarchies. Bond crossings between highly coarse-grained soft particles are prevented by applying an entanglement algorithm. We first test our method on a virtual branch point inside a linear chain to make sure it works effectively when linking two linear arms. Next, we apply our method to study the diffusive and rheological behaviors of a melt of three-armed stars. We find that the diffusive behavior of the three-armed star is very close to that of a linear polymer with the same molecular weight, while its rheological properties are close to those of a linear chain with molecular mass equal to that of the longest linear sub-chain in the star.

The origin of flow-induced alignment of spherical colloids in shear-thinning viscoelastic fluids.

We have studied the poorly understood process of flow-induced structure formation by colloids suspended in shear-thinning fluids. These viscoelastic fluids contain long flexible chains whose entanglements appear and disappear continuously as a result of brownian motion and the applied shear flow. Responsive particle dynamics simulates each chain as a single smooth brownian particle, with slowly evolving inter-particle degrees of freedom accounting for the entanglements. The colloids mixed homogeneously in all simulated quiescent dispersions and they remain dispersed under slow shear flow. Beyond a critical shear rate, which varies depending on the fluid, the colloids aggregate and form flow-aligned strings in the bulk of the fluid. In this work we explore the physical origins of this hitherto unexplained ordering phenomena, both by systematically varying the parameters of the simulated fluids and by analyzing the flow-induced effective colloidal interactions. We also present an expression for the critical shear rate of the studied fluids.

Alignment of particles in sheared viscoelastic fluids.

We investigate the shear-induced structure formation of colloidal particles dissolved in non-Newtonian fluids by means of computer simulations. The two investigated visco-elastic fluids are a semi-dilute polymer solution and a worm-like micellar solution. Both shear-thinning fluids contain long flexible chains whose entanglements appear and disappear continually as a result of Brownian motion and the applied shear flow. To reach sufficiently large time and length scales in three-dimensional simulations with up to 96 spherical colloids, we employ the responsive particle dynamics simulation method of modeling each chain as a single soft Brownian particle with slowly evolving inter-particle degrees of freedom accounting for the entanglements. Parameters in the model are chosen such that the simulated rheological properties of the fluids, i.e., the storage and loss moduli and the shear viscosities, are in reasonable agreement with experimental values. Spherical colloids dispersed in both quiescent fluids mix homogeneously. Under shear flow, however, the colloids in the micellar solution align to form strings in the flow direction, whereas the colloids in the polymer solution remain randomly distributed. These observations agree with recent experimental studies of colloids in the bulk of these two liquids.

Microphase separation and liquid-crystalline ordering of rod-coil copolymers.

Microphase separation and liquid-crystalline ordering in diblock and triblock rod-coil copolymers (with rod-to-coil fraction f=0.5) were investigated using the dissipative particle dynamics method. When the isotropic disordered phases of these systems were cooled down below their order-disorder transition temperatures T(ODT), lamellar structures were observed. For rod-coil diblock copolymers, the lamellar layers were obtained below T=2.0. This temperature was found to be higher than the T(ODT) for normal coil-coil diblock copolymers. Significant ordering of the rods was observed only below T=0.9 which is the isotropic-nematic transition temperature for rodlike fluids. For the triblock rod-coil copolymers, both microphase separation and rod ordering occurred at T=0.9. Normal coil-coil triblock copolymers were found to undergo microphase separation at T=0.8, which is about half the T(ODT) of the normal diblock copolymers. Investigations of the mean square displacement and the parallel and the perpendicular components of the spatial distribution function revealed that at low temperatures, the rod-coil diblock copolymers exhibit smectic-A and crystalline phases, while the triblock copolymers show smectic-C and crystalline phases. No nematic phases were observed at the density and interaction parameters used in this study.

Spinodal decomposition of asymmetric binary fluids in a micro-Couette geometry simulated with molecular dynamics.

The spinodal decomposition of quenched polymer/solvent and liquid-crystal/solvent mixtures in a miniature Taylor-Couette cell has been simulated by molecular dynamics. Three stacking motifs, each reflecting the geometry and symmetry of the cell, are most abundant among the fully phase separated stationary states. At zero or low angular velocity of the inner cylindrical drum, the two segregated domains have a clear preference for the stacking with the lowest free energy and hence the smallest total interfacial tension. For high shear rates, the steady state appears to be determined by a minimum dissipation mechanism, i.e., the mixtures are likely to evolve into the stacking demanding the least mechanical power by the rotating wall. The partial slip at the polymer-solvent interfaces then gives rise to a new pattern: A stack of three concentric cylindrical shells with the viscous polymer layer sandwiched between two solvent layers. Neither of these mechanisms can explain all simulation results, as the separating mixture easily becomes kinetically trapped in a long-lived suboptimal configuration. The phase separation process is observed to proceed faster under shear than in a quiescent mixture.

Finite system size effects in the interfacial dynamics of binary liquid films.

We study the relaxation dynamics of capillary waves in the interface between two confined liquid layers by means of molecular dynamics simulations. We measure the autocorrelations of the interfacial Fourier modes and find that the finite thickness of the liquid layers leads to a marked increase of the relaxation times as compared to the case of fluid layers of infinite depth. The simulation results are in good agreement with a theoretical first-order perturbation derivation, which starts from the overdamped Stokes' equation. The theory also takes into account an interfacial friction, but the difference with no-slip interfacial conditions is small. When the walls are sheared, it is found that the relaxation times of modes perpendicular to the flow are unaffected. Modes along the flow direction are relatively unaffected as long as the equilibrium relaxation time is sufficiently short compared to the rate of deformation. We discuss the consequences for experiments on thin layers and on ultralow surface tension fluids, as well as computer simulations.

Domain formation and growth in spinodal decomposition of a binary fluid by molecular dynamics simulations.

The two initial stages of spinodal decomposition of a symmetric binary Lennard-Jones fluid have been simulated by molecular dynamics simulations, using a hydrodynamics-conserving thermostat. By analyzing the growth of the average domain size R(t) with time, a satisfactory agreement is found with the R(t) proportional t1/3 Lifshitz-Slyozov growth law for the early diffusion-driven stage of domain formation in a quenched homogeneous mixture. In the subsequent stage of viscous-dominated growth, the mean domain size appears to follow the linear growth law predicted by Siggia.

Destruction of murine lymphoma cells by allogeneic immune peritoneal macrophages in vitro: an ultrastructure study.

SL2 lymphoma cells from inbred DBA/2 mice were added to monolayers of peritoneal macrophages isolated from immunized inbred C57BL/10ScCr mice. Tumor cells were removed from the supernatant most rapidly during the first few hours. Once tumor cells were bound to the macrophages, however, their destruction apparently proceeded at a constant rate. Most tumor cells initially had only very small areas of contact with macrophages. The first tumor cell changes were loss of microvilli and the formation of surface blebs that ultimately detached from the cell. Subsequently, the tumor cell rapidly rounded up while the nucleus became pyknotic and the cytoplasm vacuolated; finally, the plasma membrane lost its integrity. It was only in this stage that the macrophages actively phagocytized the tumor cells. The recorded extracellular killing of tumor cells, followed by phagocytosis of their remnants, was compared with the widely divergent descriptions of macrophage-tumor cell interaction in the literature.

Destruction of murine lymphoma cells by allogeneic peritoneal macrophages in vitro: influence of antiserum.

Peritoneal macrophages isolated from intact inbred C57BL/10ScCr mice and from inbred C57BL/10ScCr mice previously immunized with the allogeneic SL2 lymphoma were incubated with SL2 cells with and without antiserum. The mode of interaction of the cells during the first 4 hours was studied ultrastructurally. Normal macrophages interacted with the tumor cells only when antiserum was present. The main feature was a partial enveloping of the tumor cells by thin lamellipodia without any visible cell damage. Immune macrophages without antiserum killed the tumor cells extracellularly by a process of apoptosis and subsequently phagocytized the cell remnants, as described previously. In the presence of antiserum, immune macrophages phagocytized intact tumor cells with very long, slender lamellipodia. Subsequently, the tumor cells underwent intracellular necrosis that seemed to be initiated by the release by the macrophage of complete lysosomes into the phagosome containing the tumor cell. In addition to altering the mode of interaction, antiserum greatly increased the degree of cytotoxicity.

Therapy with antibody-coated immune and hyperimmune peritoneal cells in a murine lymphoma system.

Goat-antimouse (DBA/2) SL2 lymphoma immunoglobulin was specifically cytotoxic to DBA/2-derived sl2 lymphoma cells. This anti-SL2 immunoglobulin, or a part of it, was cytophylic for peritoneal macrophages as shown by target cells adhering to macrophages incubated in vitro with the immunoglobulin. The target cells became free in the medium after incubation with 0.1% trypsin for 1 hour. In in vivo experiments, the incubation of immune or hyperimmune peritoneal cells with immunoglobulin from normal goat serum or from goat-anti-SL2 serum before use in immunotherapy decreased the number of DBA/2 mice surviving for more than 35 days an intraperitoneal injection with SL2 cells compared to the number of survivors inoculated with immune or hyperimmune cells only. These results show that, in immunotherapy with immune cells, we must consider the possibility that specific antitumor antibodies and antibodies not directed against the tumor cloud the therapeutic potnecy of the immune cells.

Treatment of ocular squamous cell carcinomas in cattle with interleukin-2.

In total, 174 bovine ocular squamous cell carcinomas of varying sizes (20 to 2800 mm(2) in area) were treated daily with peritumoural injections of solvent, or solvent containing 5000 U, 20,000 U, 200,000 U, 500,000 U, 1 million U or 2 million U interleukin-2 (IL-2) for 10 days. The tumours were measured and clinically staged before treatment and at one, three, four, nine and 20 months after treatment. After 20 months, 14 per cent of the tumours treated with the solvent had regressed completely, a significantly smaller proportion than the 55 per cent treated with 5000 U IL-2, 52 per cent treated with 20,000 U IL-2, 58 per cent treated with 200,000 U IL-2, 50 per cent treated with 500,000 U IL-2, 69 per cent of tumours treated with 1 million U IL-2, 52 per cent treated with 2 million U IL-2. The tumours on the third eyelid and limbus were the most responsive.

Immunotherapy of mice with a large burden of disseminated lymphoma with low-dose interleukin 2.

Successful immunotherapy with recombinant interleukin 2 (rIL-2) of mice bearing a large burden of lymphokine-activated killer-resistant disseminated SL2 lymphoma is described. When mice were challenged i.p. with 2 x 10(4) SL2 cells on day 0 and treated with daily i.p. injections of 5,000 units rIL-2 on days 3-7, no therapeutic effect was observed. However after treatment with daily IL-2 injections on day 10-14, 25% of the mice survived. Ten days after this tumor challenge more than 10(8) SL2 cells were present growing as ascitic tumor. On day 10, SL2 cells were also present as solid tumor in the greater omentum and as metastases in lungs and liver. Surviving mice were able to reject a second challenge with SL2 cells given on day 60. A second challenge with P815, another DBA/2 tumor, resulted in death of the mice due to tumor development. This finding is of particular importance as the SL2 cells are resistant to lymphokine-activated killer activity. Thus local (i.p.) injection of low dose rIL-2 can cause the systemic rejection of advanced and metastasized cancer. Our data indicate that IL-2 can strongly enhance a specific immune reaction against tumor cells.

Insulin-like growth factor gene expression in human smooth muscle tumors.

A role for insulin-like growth factors (IGF) in autocrine or paracrine growth stimulation of tumor cells has been proposed for tumors of different origins. We have studied IGF gene expression in human uterus smooth muscle (myometrium) and in a panel of benign (leiomyoma) and malignant (leiomyosarcoma) smooth muscle tumors. Using RNA transfer blot analysis we could demonstrate that in smooth muscle tissue and tumors IGF genes are differentially expressed. The mRNA species detected had the same size as reported for IGF mRNAs from other tissues. However, the abundance of the IGF gene transcripts varied from tissue to tissue. The amounts of IGF mRNAs detected in smooth muscle tumors were compared to the levels found in normal smooth muscle. The IGF-I gene was expressed at high levels in normal myometrium and in leiomyomas but appears to be repressed in leiomyosarcomas. Also the IGF-I peptide was detected in myometrium and in leiomyomas, but in leiomyosarcomas the level was substantially lower. The IGF-II gene was expressed at low levels in normal myometrium and leiomyomas but is activated in leiomyosarcomas. With increasing malignancy from the two major IGF-II mRNA species, 6.0 and 4.8 kilobases, in particular the 6.0-kilobase mRNA is produced at higher levels. In conclusion, these data suggest that for IGF-I a role in tumor cell growth is not likely, but probably IGF-II is involved in malignant smooth muscle tumor growth progression.

An avaII restriction fragment length polymorphism in the insulin-like growth factor II gene and the occurrence of smooth muscle tumors.

To detect a previously described AvaII restriction fragment length polymorphism (RFLP) in the human insulin-like growth factor II (IGF-II) gene we used the polymerase chain reaction (PCR) and genomic sequencing. The RFLP is located in exon 9 of the IGF-II gene at nucleotide 820 (GenBank accession number X07868) as a C-->T transition. Digestion with AvaII reveals a two-allele polymorphism, an a allele in which the AvaII site is not present, and a b allele. In healthy Dutch persons (n = 26), the frequency of the a allele was 62%. A similar a allele frequency was found in groups of Japanese (53%, n = 65) and Chinese (54%, n = 84), while in a French group the frequency was significantly lower (25%, n = 52). In Dutch individuals that had developed benign (n = 11; all women) and malignant (n = 9; 2 women and 7 men) smooth muscle tumors, a significantly higher frequency of 83% for the a allele was found. Since there was no difference between the presence of the a and b alleles in normal and tumor tissue of the same individual, the higher a allele frequency was not due to mutation in the IGF-II gene or loss of heterozygosity. There was no correlation between the presence of the a allele and expression of the IGF-II gene. The data reveal a correlation between homozygosity for the a allele and the occurrence of smooth muscle tumors. Women homozygous for the IGF-II a allele are more prone to develop a leiomyoma than women who are heterozygous or homozygous for the b allele. Furthermore, in both women and men the risk for leiomyosarcomas seems to be higher in a allele homozygotes.

Expression and CpG methylation of the insulin-like growth factor II gene in human smooth muscle tumors.

Previously we have shown that expression of the insulin-like growth factor II (IGF-II) gene in 36 normal smooth muscle tissues (myometria) and 26 benign smooth muscle tumors (leiomyomas) was detectable by Northern blot analysis but that the RNA levels were low. In 9 of 20 malignant smooth muscle tumors (leiomyosarcomas) IGF-II gene expression was also low or absent, while in 11 of 20 the IGF-II gene was abundantly expressed. In 32 of these tissues we have now studied the DNA methylation state of the IGF-II gene. For the analysis of overall methylation of the gene the restriction endonucleases HpaII and MspI were used. In normal smooth muscle and in leiomyomas the IGF-II gene appeared to be methylated. In leiomyosarcomas with low IGF-II gene expression the DNA was partly demethylated. In leiomyosarcomas with abundant IGF-II gene expression overall methylation of the DNA tended to be low. In addition, we have studied the methylation state of one particular CpG site in the IGF-II gene with the restriction endonuclease AvaII. The results of the latter analysis confirm the analysis with HpaII and MspI. In conclusion, in malignant smooth muscle tumors the data indicate an inverse correlation between CpG methylation and expression of the IGF-II gene.

Modifying self-assembly and species separation in three-dimensional systems of shape-anisotropic particles.

The behaviors of large, dynamic assemblies of macroscopic particles are of direct relevance to geophysical and industrial processes and may also be used as easily studied analogs to micro- or nano-scale systems, or model systems for microbiological, zoological, and even anthropological phenomena. We study vibrated mixtures of elongated particles, demonstrating that the inclusion of differing particle "species" may profoundly alter a system's dynamics and physical structure in various diverse manners. The phase behavior observed suggests that our system, despite its athermal nature, obeys a minimum free energy principle analogous to that observed for thermodynamic systems. We demonstrate that systems of exclusively spherical objects, which form the basis of numerous theoretical frameworks in many scientific disciplines, represent only a narrow region of a wide, multidimensional phase space. Thus, our results raise significant questions as to whether such models can accurately describe the behaviors of systems outside this highly specialized case.