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Objective. To determine the level of expression and clinical importance of serum miR-204 and miR-451 in patients with pulmonary arterial hypertension caused by congenital heart disease (CHD-PAH). Methods. From July 2019 to January 2021, 114 infants with congenital heart disease (CHD) were hospitalized at Qingdao’s Fuwai Cardiovascular Hospital. They were grouped into categories: CHD (53 cases) and CHD-PAH (61 cases) based on whether they had pulmonary hypertension (PAH). In addition, 60 healthy children were selected as the control group. All children underwent routine biochemical examination, echocardiography, and pulmonary arterial pressure examination. By using an enzyme-linked immunosorbent assay (ELISA), the levels of brain natriuretic peptide (BNP) and asymmetric dimethylarginine (ADMA) in the blood were measured. Additionally, reverse transcription-polymerase chain reaction (RT-PCR) was used to determine the expression levels of miR-204 and miR-451 in peripheral blood. The correlation between miR-204, miR-451, BNP, ADMA, and mPAP was investigated using Pearson correlation analysis. Results. Consequently, the TC, BNP, and ADMA serum levels were considerably higher in the CHD and CHD-PAH groups than in the control group ( P < 0.05 ), whereas BNP and ADMA serum levels were significantly higher in the CHD-PAH group than in the CHD group ( P < 0.05 ). According to RT-PCR data, the expression levels of miR-204 and miR-451 in the peripheral blood of children in the CHD and CHD-PAH groups were significantly lower ( P < 0.05 ) when compared to the control group. The expression levels of miR-204 and miR-451 in the peripheral blood of children in the CHD-PAH group were substantially lower ( P < 0.05 ) than in the CHD group. Significantly, the findings of the ROC curve revealed that the area under the curve (AUC) of CHD-PAH diagnosed by miR-204 and miR-451 alone was 0.737 and 0.725, respectively, and the AUC of joint diagnosis was 0.840, which was greater than that of single diagnosis ( P < 0.05 ). Patients with CHD-PAH had lower levels of miR-204 and miR-451 in their blood, and this was found to be associated with BNP, ADMA, and mPAP, analyzed by Pearson correlation. Conclusions. Children with CHD-PAH can be diagnosed based on aberrant expressions of miR-204 and miR-451 in their peripheral blood serum. Moreover, CHD-PAH can be diagnosed if the combination of miR-204 and miR-451 is detected as a biomarker, which has a higher diagnostic value.

Understanding the mesoscale structure and regime transition in bubble columns is of great significance for reactor design and scaleup. Based on the energy-minimization multiscale (EMMS) model, a noncooperative game model with constraints is proposed to investigate the structural properties of gas–liquid systems in which small and large bubbles are chosen as players and the energy consumption form the objective function. The conservation equations of the system can be regarded as the constraints of the game. For the formulated noncooperative game model, the concept of the generalized Nash equilibrium (GNE) is used to characterize the solution. An algorithm is developed to numerically compute the GNE and some important structural parameters in the system. The numerical results show the existence of the GNE for all values of the superficial gas velocity Ug. As Ug varies, the trends in the state variables can be observed and the critical point of Ug identified. The overall trend of the flow regime transition agrees with the original EMMS model and experimental results, although the GNE calculation also reveals different single-bubble dominant mechanisms with increasing Ug.

Droplet size distribution (DSD) of an emulsion is one of the important properties that affect product quality, stability, appearance, and rheology. Reliable prediction of DSDs in high-pressure homogenizers under various emulsion formulation and processing conditions is still challenging. A zero-dimensional population balance equation (PBE) model was developed to take account of the heterogeneous turbulent dissipation rate through its volume fraction distribution rather than the mean value as the coupling parameter between CFD and PBE. Furthermore, the effect of emulsifier on coalescence was also incorporated through the surface coverage of emulsifier. The model can give satisfactory DSDs under different oil fractions, homogenization pressures, and emulsifier concentrations for the oil-in-water emulsions. Moreover, the predictions were more sensitive to the breakage kernel parameters than to the coalescence ones or the number of daughter droplets. This approach is beneficial to the fast computational product design with less computational cost and reasonable prediction.

Multiple breakup refers to a sequence of events through which a single droplet eventually produces multiple daughter droplets at a given flow condition. It is a more common phenomenon than binary breakup assumed in the existing breakage kernel models. Using phase-field lattice Boltzmann method, this work investigates the effects of Reynolds number, capillary number and soluble surfactant on multiple breakup in shear flow. We find that the critical capillary number in multiple breakup decreases as Reynolds number increases, and the regime map for a surfactant-free system could be classified into the non-breakup, elementary breakup, multiple breakup, filament and coalescence regimes. In the system of surfactants, the multiple breakup regime widens and the coalescence regime narrows. A correlation is then proposed to predict the number of breakup events and daughter droplets at a confinement ratio of 0.5. This may provide some clues on developing new breakage kernels in population balance modeling.

We report an approach to synthesize silver nanowires by diffusion limitation. Silver particles are synthesized by electrochemical reaction in mixtures of silver nitrate solution and glycerol. Without glycerol, the main product is silver dendrites. With the addition of glycerol, silver nanowires are instead produced in abundance. The addition of glycerol increases the viscosity of the solvent, which restricts the diffusion of silver ions. Under conditions of diffusion limitation, a rapid electrochemical reaction leads to the formation of a chemical concentration gradient at the growth front of the nuclei, which results in the formation of silver dendritic structures. The increase of solvent viscosity attenuates the concentration gradient, which limits secondary nucleation on the sides of the silver rods, leading to the formation of 1D silver nanowires instead of dendrites. An increase of silver salt concentration sharpens the concentration gradient, which leads to the formation of dendritic structures again, confirming the dominant role of the interface chemical distribution in the structural evolution of the product materials. The silver nanowires synthesized are used to fabricate a conductive film by vacuum filtration followed by fixing and welding processes. The film shows high conductivity and excellent flexibility.

Precise and rational control of droplet size distribution (DSD) is important in emulsification for target-oriented product design. To develop a complete DSD model, crossing the two mesoscales of two different levels is of great significance, viz., the emulsifier adsorption at interfacial level (Mesoscale 1) and the droplet breakage and coalescence in turbulence in rotor-stator device level (Mesoscale 2). While the first mesoscale can be simulated by coarse-grained molecular dynamic (CGMD), the second has been investigated in computational fluid dynamics and population balance model (CFD-PBM) simulation through the Energy-Minimization Multi-Scale (EMMS) approach in Part I. We then developed a model framework in Part II, coupling CGMD and CFD-PBM simulation through surfactant transport equations in bulk phase and at interface, with source terms taking account of emulsifier adsorption parameters. The parameters including maximal adsorption amount, diffusion coefficient and adsorption/desorption kinetic constants are acquired from CGMD. The coalescence efficiency is then corrected by the interfacial area fraction not occupied by surfactant and fed into the coalescence kernel functions in PBM. Compared to traditional CFD-PBM simulation, the coupled model can greatly improve the simulation of DSD, Sauter mean diameter, median diameter and span for high dispersed phase amount (DPA), and correctly reflect the influence of DPA, surfactant concentration and rotational speed of rotor-stator (RS) devices. While the simulation cases validate and demonstrate the advantage of this new model framework, it is also promising to incorporate different types of surfactant in future.

Fractal tree-like microchannel is advantageous to faster emulsification and droplet production in microchannels. Although computer simulation is becoming a powerful tool for design and optimization, precise treatment of a large number of walls in fractal tree-like microchannel is troublesome. The numerical roughness caused by traditional bounce-back boundary condition may accumulate and generate large errors in flow pattern recognization. In this paper, an approach integrating Immersed Boundary Method (IBM), Phase-Field Model (PFM) and Lattice Boltzmann Method (LBM) is developed, aiming to accurately simulate droplet splitting in fractal tree-like microchannel system involving surfactants and wetting boundary walls. Then the operating conditions including capillary number, flow rate ratio and wetting boundary conditions were optimized. We found that the capillary number in 0.02 ∼ 0.05 and flow rate ratio in 1/3.6 ∼ 1/9 can accelerate emulsification. Hydrophobic-lipophilic walls generate a slug-like water drop of bullet shape and thin oil film between the drop and walls, facilitating drop movement in microchannel. The effects of surfactant, Marangoni stress and interfacial tension force on droplet splitting were investigated. The contact walls at the corner and forks resist the surfactant migration, and therefore enrich surfactant at the upstream end of drop interface, leading to uneven distribution of interfacial tension. The breaking interfaces and the interfaces contacting with wall or fork are subjected to opposite reaction of Marangoni stress and tend to be thinner and finally deform or break.

Effects of internals on the fluid dynamics and reaction performance in a pilot-scale slurry bubble column reactor (dc = 0.29 m, h = 4 m) for Fischer-Tropsch synthesis were investigated by CFD simulation in two stages: the first stage with only fluid flow and the second stage coupled with flow, mass transfer and reaction. At the same superficial gas velocity based on free area of column cross-section, the effects of internals on hydrodynamics, such as gas holdup, slurry large-scale recirculation and turbulent viscosity, were analyzed in the first stage. Considering the uncertainty of available mass transfer models, these models were evaluated first. These models differ in magnitude of volumetric mass transfer coefficient and relative mass transfer capacity of H2 to CO. The simulations indicated that, for the slurry column reactor simulated in this paper, the reaction performances simulated with different mass transfer models are similar, and the reactor is in the reaction-limited regime at the relatively low solid holdup condition. The presence of internals decreases gas holdup and reactant concentrations in the slurry phase but increases the mass transfer and reaction rates. Moreover, the syngas conversion and reactor productivity per unit volume are obviously enhanced with the increase of the number of internals.

Large-bubbles and small-bubbles represent two competing dominant structures in bubble columns. Understanding the structure evolution is of great significance in physical modeling. Dynamic gas disengagement (DGD) and dual conductivity probe (DCP) methods were applied to demarcate large and small-bubbles. While the number-based BSD only presents a single peak, the dual peaks could only be found from the volume-based BSD. The demarcation method based on the holdup ratio of the two bubble groups proves more reasonable to reflect the intrinsic nature of bubble structure. A sharp change of gas holdup was detected for the uniform distributor. The difference in gas holdup structure of coarse and uniform distributors was contributed mainly by small-bubbles at homogeneous regime, yet by large-bubbles at transition and heterogeneous regimes. However, the coexistence of small and large-bubbles is the intrinsic nature, and the effect of gas distributor is marginal in heterogeneous regime.

Drag model is of critical importance for CFD simulation of flow characteristics in bubble columns. In this study, a novel drag model termed DBS-Local for the ratio of effective drag coefficient to bubble diameter is derived from the Dual-Bubble-Size (DBS) model based on Energy-Minimization Multi-Scale (EMMS). A thorough comparison demonstrates that without adjustable parameters, both the DBS-Local model and the DBS-Global model are capable of improving the prediction of total and local gas holdup as well as the flow characteristics in different flow regimes. By contrast, other drag models either overestimate or underestimate the total or local gas holdup in certain flow regimes. Moreover, the centerline liquid velocity predicted by DBS-Local model shows best agreement with experiments, whereas the other models, especially the Ishii-Zuber model, over-estimate liquid velocity. This study demonstrates the significance of modeling the bubble-swarm effect based on EMMS approach.

Bubble properties, such as local gas holdup, bubble frequency, bubble velocity, bubble size, and interfacial area concentration, are of great significance for the design and scale-up of bubble columns. In the present study, bubble properties in a 0.15 m in diameter bubble column were measured by a dual-tip conductivity probe. A new method was proposed to establish the relationship between chord length distribution (CLD) and bubble size distribution (BSD) without the assumption of fixed bubble aspect ratio. The bubble velocity and BSD measured by the probe were validated by a high speed camera in a square column. The results indicate that considering the decreasing of bubble aspect ratio with bubble diameter is required to obtain accurate BSD from measured CLD. Furthermore, the measured bubble properties at different axial positions show that the flow development is much more rapid in the churn-turbulent regime than that in the homogeneous regime. It is also found that the bubble breakage is dominant over the bubble coalescence in the homogeneous regime due to the large bubbles generated at the perforated distributor. The measured results provide a benchmark data for validation of CFD simulation.

In the present study, influence of interfacial forces, including drag force and lateral forces (lift force, turbulent dispersion force and wall force) on the hydrodynamics in pilot-scale bubble columns with internals is analyzed. The results indicate that the lateral forces may be optional for the hollow columns, but they are required to accurately predict flow characteristics in the bubble columns with internals. Furthermore, it has been found that the bubbly flow behavior in the bubble columns with internals is more sensitive to the lateral forces in comparison with those without internals, and the complex geometry significantly alters the response of bubbly flow to the interfacial forces. In addition, despite the insignificant effect on gas holdup, the presence of internals gives rise to an enhancement of large-scale liquid circulation due to the remarkable decrease of turbulent viscosity.

The mechanism of the meso-scale structure in the gas-liquid system in bubble columns is of great significance in chemical engineering. Based on the multi-objective problem indwelling the EMMS(energy-minimization-multi-scale) model, we studied the problem from a novel point of the noncooperative game theory. In our previous works, we formulated the problem as a game between different dominant mechanisms of particles and the conservation equations of system state variables were considered as constraints. Then we chose several free state variables as strategies for each player for some reason. The system state at GNE(generalized Nash equilibrium) seems quite coincident with the model prediction and the experimental results in the literature. In this paper, we will choose different free variables as strategies of the players and thus will build a different game model for the same problem. Then we will see different GNE results about the steady states as well as the important parameters of the system, which implies that the problem is not so simple or naive as we regarded before. Actually it involves much tough and fundamental issues that we must consider very carefully.

Multiple breakup refers to a sequence of events through which a single droplet eventually produces multiple daughter droplets in a shear flow. It is a more common phenomenon than binary breakup. Using a phase-field lattice Boltzmann method, this work investigates the effects of Reynolds number, capillary number and soluble surfactant on multiple breakup in shear flow. We find that the regime map for a droplet in a surfactant-free shear flow can be segmented into the non-breakup, elementary breakup, multiple breakup, filament and coalescence regimes. By contrast, the multiple-breakup regime widens and the coalescence regime narrows in the system of surfactant. This difference on macroscale regimes originates from the mesoscale behaviour caused by the interaction of surfactant and shear flow. While surfactant hinders droplet breakup at lower Re, it promotes breakup at higher Re. The interfaces around the breaking points are stretched by the opposite reaction of Marangoni stress. While the regime map gives the number of daughter droplets in a given shear flow, a correlation is proposed to calculate the composition of breakup events, viz. the number of binary breakup and of ternary breakup events.

Experiments and CFD-PBM simulation were performed in this study to investigate the effects of helically-finned tube (HFT) internals on the gas-liquid flow in a laboratory-scale bubble column. Hydrodynamic parameters, including gas holdup, bubble size and flow field as well as turbulence properties, were compared for an empty column and other two columns with bare-tube (BT) or HFT internals. We found that bare rods promote the stability of two-phase flow, whereas helical fins destabilize the flow. Meanwhile, the spatial distribution of gas holdup or bubble size becomes more uniform in the presence of HFT internals, whereas liquid velocity or turbulent dissipation rate evidently decreases. The fin-induced spiral movement, flow resistance and bubble accumulation are the mechanisms underlying these experimental observations. Integration of rod and helical fin may be promising for process intensification in bubble columns.

Experiments and simulations were conducted for bubble columns with diameter of 0.2 m (180 mm i.d.), 0.5 m (476 mm i.d.) and 0.8 m (760 mm i.d.) at high superficial gas velocities (0.12–0.62 m·s− 1) and high solid concentrations (0–30 vol%). Radial profiles of time-averaged gas holdup, axial liquid velocity, and turbulent kinetic energy were measured by using in-house developed conductivity probes and Pavlov tubes. Effects of column diameter, superficial gas velocity, and solid concentration were investigated in a wide range of operating conditions. Experimental results indicated that the average gas holdup remarkably increases with superficial gas velocity, and the radial profiles of investigated flow properties become steeper at high superficial gas velocities. The axial liquid velocities significantly increase with the growth of the column size, whereas the gas holdup was slightly affected. The presence of solid in bubble columns would inhibit the breakage of bubbles, which results in an increase in bubble rise velocity and a decrease in gas holdup, but time-averaged axial liquid velocities remain almost the same as that of the hollow column. Furthermore, a 2-D axisymmetric k–e model was used to simulate heterogeneous bubbly flow using commercial code FLUENT 6.2. The lateral lift force and the turbulent diffusion force were introduced for the determination of gas holdup profiles and the effects of solid concentration were considered as the variation of average bubble diameter in the model. Results predicted by the CFD simulation showed good agreement with experimental data.

Prediction of gas holdup and bubble size distribution is of paramount importance for modeling mass and heat transfer and reactions in multiphase reactors. Addition of hydrophilic particles into gas-liquid systems usually decreases gas holdup and increases bubble size. Rational prediction is still difficult in view of the complex effects of solid particles on gas-liquid flows, e.g., the increase of slurry apparent density and viscosity, the reduced film drainage time during bubble coalescence, and the attenuation of liquid turbulent dissipation rate. This work attempts to construct a rational step-by-step approach to analyze these particle effects in CFD-PBM modeling. First, the EMMS-PBM approach is used to improve the BSD simulation in a solid-free system. Then for a gas-slurry system, we propose a correlation of attenuation factor for turbulent dissipation rate in PBM kernel models. Combining EMMS-PBM and the attenuation factor can well predict the gas holdup and BSD in slurry bubble columns.

Drag force plays important roles in the prediction of gas-liquid flow in stirred tanks, but systematic studies on drag models are still lacking. In this study, we evaluated the effect of four different drag models (DBS-Local, DBS-Global, Brucato, Schiller-Naumann) in which the DBS model was originally developed for bubble columns. The flow regime, liquid flow field and gas holdup predicted by the four drag models are compared with experiments. The DBS-Local and DBS-Global models show the advantages over the other two in the prediction of gas dispersion and flow structure, since the ratio of effective drag coefficient to bubble diameter is either one or two-orders of magnitude over-estimated by the Brucato model or under-predicted by the Schiller-Naumann model. The flow regime at different rotational speeds and the effects of gas dispersion on the liquid loop structure can be correctly predicted by the DBS drag models. This study demonstrates that the drag model is extremely important to accurately predict the gas-liquid flow in stirred tanks.

A dual-scale turbulence model is applied to simulate cocurrent upward gas–liquid bubbly flows and validated with available experimental data. In the model, liquid phase turbulence is split into shear-induced and bubble-induced turbulence. Single-phase standard k - e model is used to compute shear-induced turbulence and another transport equation is added to model bubble-induced turbulence. In the latter transport equation, energy loss due to interface drag is the production term, and the characteristic length of bubble-induced turbulence, simply the bubble diameter in this work, is introduced to model the dissipation term. The simulated results agree well with experimental data of the test cases and it is demonstrated that the proposed dual-scale turbulence model outperforms other models. Analysis of the predicted turbulence shows that the main part of turbulent kinetic energy is the bubble-induced one while the shear-induced turbulent viscosity predominates within turbulent viscosity, especially at the pipe center. The underlying reason is the apparently different scales for the two kinds of turbulence production mechanisms: the shear-induced turbulence is on the scale of the whole pipe while the bubble-induced turbulence is on the scale of bubble diameter. Therefore, the model reflects the multi-scale phenomenon involved in gas–liquid bubbly flows.

Hydrodynamics in a 0.8 m diameter bubble column with pin-fin tube internals covering 9.2% total cross-sectional area of the column is experimentally investigated. The experimental results indicated that pin-fin tube internals significantly affect gas holdup and liquid velocity. It can be inferred from the measured data that the pin fins on the tubes obviously reduce the distributor region in the column. Furthermore, non-uniform arrangement of pin-fin tube internals through symmetrically removing two tubes near column wall gives rise to severe gas short-circuiting and even no downward liquid flow there, while there is imperceptible influence for bubble column with non-uniform plain tube internals.

3D Eulerian-Eulerian model was applied to simulate the gas-liquid two-phase flow in a stirred tank of dual Rushton turbines using computational fluid dynamics (CFD). The effects of two different bubble treatment methods (constant bubble sizevs. population balance model, PBM) and two different coalescence models (Luo modelvs. Zaichik model) on the prediction of liquid flow field, local gas holdup or bubble size distribution were studied. The results indicate that there is less difference between the predictions of liquid flow field and gas holdup using the above models, and the use of PBM did not show any advantage over the constant bubble size model under lower gas holdup. However, bubble treatment methods have great influence on the local gas holdup under larger gas flow rate. All the models could reasonably predict the gas holdup distribution in the tank operated at a low aeration rate except the region far from the shaft. Different coalescence models have great influence on the prediction of bubble size distribution (BSD). Both the Luo model and Zaichik model could qualitatively estimate the BSD, showing the turning points near the impellers along the height, but the quantitative agreement with experiments is not achieved. The former over-predicts the BSD and the latter under-predicts, showing that the existing PBM models need to be further developed to incorporate more physics.

Numerical simulation of bubble dynamics in bubble columns with internals is investigated, and the results show that internal solid walls significantly affect bubble behavior. The trajectories of the selected diameter bubbles tend to be rectilinear, and there are rocking and oscillation for small bubbles which are associated with vortex shedding in the bubble wake. Bubble axially elongates and bubble aspect ratio increases with the increase of internals covered cross-sectional area (CSA). Simultaneously, bubble rise velocity is reduced, and it is related with internals covered CSA and pitch type. Moreover, simulation of bubble dynamics in different diameter internals indicates that hydraulic diameter is the key parameter for scale-up of bubble columns with internals. In addition, bubble breakup and turbulence structure are profoundly impacted by the internal solid walls.

The dynamic mechanical properties of a well-characterized series of homogeneous ethylene/1-octene copolymers with different random hexyl branch contents and prepared using different cooling conditions have been examined using dynamic mechanical analysis (DMA). It was confirmed that the relaxation behavior of copolymers varied continuously with the branch content: the magnitude of the β relaxation increased with branch content while the intensity of the α relaxation decreased with the branch content; both relaxation temperatures decreasing with increasing branch level in the copolymers. Copolymers prepared at different cooling conditions were further examined and strikingly continuous changes were found for the first time. The β relaxation was shown to correlate to the amorphous region, while the α1 and α2 relaxations can be clearly differentiated for some samples and are assumed to be associated with the interlamellar slip and intra-crystalline c-shear processes respectively. With increasing cooling rate, the relative intensity of α1 relaxation to α2 relaxation was found to decrease while the β relaxation did not change. The most informative data is determined from deconvolution of tan δ spectra. In higher crystallinity polymers the α1 and α2 relaxations are closely related in activation energy but have different temperature locations. For lower crystallinity systems, where the α1 relaxation cannot be observed, the α2 and β relaxations are closely linked, with activation energies approaching one another. These results show very clearly that, although the observed relaxation data can be separated through deconvolution into three separate peaks, the behaviors are closely linked. Presumably, this a clear reflection of the role of tie molecules in binding phases together and in influencing dynamic mechanical behavior. A clear change of behavior has also been observed in the β relaxation when a distinct amorphous phase exists outside of the spherulites, confirming the general belief that the crystalline phase influences the amorphous phase when it is confined within a spherulite. Again, this behavior is reflecting the role of tie molecules in binding together the nanocomposite structure of a spherulite.

Rare earth ions doped glasses producing visible upconversion emissions are of great interest due to their potential applications in the photonics filed. In fact, practical application of upconversion emissions has been used to obtain color image displays and white light sources. However, there are few reports on the thermal effect on tuning the emission color of the RE doped materials. In this work, the Er 3+ /Tm 3+ /Yb 3+ tridoped oxyfluoride glasses were prepared through high temperature solid-state method. Under a 980 nm diode laser excitation, the upconversion emissions from the samples were studied. At room-temperature, bright white luminescence, whose CIE chromaticity coordinate was about (0.28, 0.31), can be obtained when the excitation power was 120 mW. The emission color was changed by varying the intensity ratios between RGB bands, which are strongly dependent on the rare earth ions concentration. The temperature dependent color emissions were also investigated. As temperature increased, the intensities for the emission bands presented different decay rates, finally resulting in the changing of the CIE coordinate. When the temperature was 573 K, white light with color coordinate of (0.31, 0.33) was achieved, which matches well with the white reference (0.33, 0.33). The color tunability, high quality of white light and intense emission intensity make the transparent oxyfluoride glasses excellent candidates for applications in solid-state lighting.

We showed previously in neocortical explants, derived from developing wild-type and estrogen receptor (ER)-alpha gene-disrupted (ERKO) mice, that both 17alpha- and 17beta-estradiol elicit the rapid and sustained phosphorylation and activation of the mitogen-activated protein kinase (MAPK) isoforms, the extracellular signal-regulated kinases ERK1 and ERK2. We proposed that the ER mediating activation of the MAPK cascade, a signaling pathway important for cell division, neuronal differentiation, and neuronal survival in the developing brain, is neither ER-alpha nor ER-beta but a novel, plasma membrane-associated, putative ER with unique properties. The data presented here provide further evidence that points strongly to the existence of a high-affinity, saturable, 3H-estradiol binding site (K(d), approximately 1.6 nm) in the plasma membrane. Unlike neocortical ER-alpha, which is intranuclear and developmentally regulated, and neocortical ER-beta, which is intranuclear and expressed throughout life, this functional, plasma membrane-associated ER, which we have designated "ER-X," is enriched in caveolar-like microdomains (CLMs) of postnatal, but not adult, wild-type and ERKO neocortical and uterine plasma membranes. We show further that ER-X is functionally distinct from ER-alpha and ER-beta, and that, like ER-alpha, it is re-expressed in the adult brain, after ischemic stroke injury. We also confirmed in a cell-free system that ER-alpha is an inhibitory regulator of ERK activation, as we showed previously in neocortical cultures. Association with CLM complexes positions ER-X uniquely to interact rapidly with kinases of the MAPK cascade and other signaling pathways, providing a novel mechanism for mediation of the influences of estrogen on neuronal differentiation, survival, and plasticity.

Confocal laser scanning microscopy was used to identify the cells within organotypic slice cultures of the developing mouse cerebral cortex that respond to estradiol treatment by phosphorylation of ERK1 and ERK2. Estrogen-responsive cells resembled neurons morphologically and expressed the neuronal marker microtubule-associated protein 2B. The intracellular distribution of the phospho-ERK signal was both cytoplasmic and nuclear, but inhibition of protein synthesis abolished the appearance of the nuclear signal. ERK1and ERK2 also coimmunoprecipitated with heat shock protein 90 (Hsp90) in the cerebral cortical explants. Geldanamycin effectively disrupted this association and prevented ERK phosphorylation. Surprisingly, MEK2 but not MEK1 was the principal mediator of estradiol-induced activation of ERK. Our data demonstrate the requirement for Hsp90 in estrogen-induced activation of ERK1 and ERK2 by MEK2 in the developing mouse cerebral cortex and also provide insight into alternative mechanisms by which estradiol may influence cytoplasmic and nuclear events in responsive neurons via the MAP kinase cascade.

In neocortical explants, E2 activates various signaling components of the MAPK cascade, including B-Raf and MAPK kinase-dependent ERK, suggesting a possible role in the differentiative actions of E2 in the brain. To further characterize the signaling pathways activated by E2, we determined whether c-Src, a member of the Src family of nonreceptor tyrosine kinases and an important modulator of both the MAPK cascade and neuronal differentiation, may play a role in E2 signaling. The present studies show for the first time in the brain that E2 elicits phosphorylation of c-Src on three functionally critical tyrosine residues (Y220, Y423, and Y534), and that this phosphorylation occurs despite disruption of ER alpha (in ER knockout mice). PP2, a Src family kinase inhibitor, suppressed not only E2-induced phosphorylation of c-Src, but ERK phosphorylation as well, suggesting that c-Src may be an upstream regulator of E2 signaling. E2-induced phosphorylation of c-Src is associated with increased tyrosine phosphorylation of Shc, increased association of Shc with Grb2, and induction of Ras, but not Rap1, activation. Together, these data provide evidence that E2 activates a novel c-Src-dependent signal transduction pathway in the developing brain.

The spinal muscular atrophies (SMAs), characterized by spinal cord motor neuron depletion, are among the most common autosomal recessive disorders. One model of SMA pathogenesis invokes an inappropriate persistence of normally occurring motor neuron apoptosis. Consistent with this hypothesis, the novel gene for neuronal apoptosis inhibitory protein (NAIP) has been mapped to the SMA region of chromosome 5q13.1 and is homologous with baculoviral apoptosis inhibitor proteins. The two first coding exons of this gene are deleted in approximately 67% of type I SMA chromosomes compared with 2% of non-SMA chromosomes. Furthermore, RT-PCR. analysis reveals internally deleted and mutated forms of the NAIP transcript in type I SMA individuals and not in unaffected individuals. These findings suggest that mutations in the NAIP locus may lead to a failure of a normally occurring inhibition of motor neuron apoptosis resulting in or contributing to the SMA phenotype.

We have shown previously in the developing cerebral cortex that estrogen elicits the rapid and sustained activation of multiple signaling proteins within the mitogen-activated protein (MAP) kinase cascade, including B-Raf and extracellular signal-regulated kinase (ERK). Using estrogen receptor (ER)-alpha gene-disrupted (ERKO) mice, we addressed the role of ER-alpha in mediating this action of estrogen in the brain. 17beta-Estradiol increased B-Raf activity and MEK (MAP kinase/ERK kinase)-dependent ERK phosphorylation in cerebral cortical explants derived from both ERKO and their wild-type littermates. The ERK response was stronger in ERKO-derived cultures but, unlike that of wild-type cultures, was not blocked by the estrogen receptor antagonist ICI 182,780. Surprisingly, both the ER-alpha selective ligand 16alpha-iodo-17beta-estradiol and the ER-beta selective ligand genistein failed to elicit ERK phosphorylation, suggesting that a different mechanism or receptor may mediate estrogen-induced ERK phosphorylation in the cerebral cortex. Interestingly, the transcriptionally inactive stereoisomer 17alpha-estradiol did elicit a strong induction of ERK phosphorylation, which, together with the inability of the ER-alpha- and ER-beta-selective ligands to elicit ERK phosphorylation, and of ICI 182,780 to block the actions of estradiol in ERKO cultures, supports the hypothesis that a novel, estradiol-sensitive and ICI-insensitive estrogen receptor may mediate 17beta-estradiol-induced activation of ERK in the brain.

Spinocerebellar ataxia type 2 (SCA2) is a neurodegenerative disease recently mapped to chromosome 12q close to the locus D12S84 by genetic linkage analysis. To generate additional genetic markers in the SCA2 region, we constructed a physical map of the region using yeast artificial chomosome (YAC), P1 artificial chromosome (PAC) and cosmid clones. The physical map was found to agree well with the genetic map. Three novel microsatellite markers were isolated and physically mapped. A novel approach to isolate CAG repeats directly from YAC DNAs is described.

We present a method for the isolation of YAC insert sequences by representational difference analysis (RDA). To achieve maximal representation of the sequences, the amplicons were generated from a Mbol digestion product. RDA was performed using a 970 kb insert YAC clone. After two rounds of re-association and selective amplification 92% of the difference product represented sequences derived from the YAC insert. Twenty insert-specific sequence-tagged sites were readily defined. The difference product was also successfully used to isolate microsatellite markers, to identify clones from a human PAC library and as a chromosome painting probe in fluorescence in situ hybridization.

The effect of the tumor promoter phorbol 12-myristate 13-acetate (PMA) on proliferation and differentiation of normal mammary epithelial cells from 50-day-old virgin rats was investigated using a model system that allows for full morphological and functional development of the cells. In this model, mammary epithelial cells are grown within a reconstituted basement membrane in a defined serum-free medium. PMA at a concentration of 10−6 M effected translocation of protein kinase C from cytosol to membrane. At the same concentration, it stimulated cell proliferation both in the presence and absence of EGF, and this stimulation was observed even when PMA exposure was limited to 15 min at the time of each media change. In contrast to the effect on proliferation, PMA at concentrations of 10−7 and 10−6 M inhibited functional differentiation as assessed by casein accumulation. Phorbol 12,13-dibutyrate at 10−6 M also stimulated proliferation and inhibited casein accumulation and was more effective than PMA in both cases. In contrast, the nonactive tumor promoter 4-α PMA had no effect on either proliferation or differentiation. One of the most striking effects of PMA was its ability to stimulate an atypical ductal morphogenesis, as manifested by the formation of intricate web-like colonies, and to inhibit the development of the well-differentiated alveolar-like multilobular colonies. PMA was also shown to completely suppress the growth of the squamous-like colonies that develop when EGF is absent or deficient. These effects of phorbol esters in mammary epithelial cells to stimulate proliferation, inhibit functional differentiation, and stimulate the development of ductal colonies are consistent with the suggestion that the signal transduction pathways evoked by PMA could act to stimulate the growth of initiated cells or render normal cells more sensitive to carcinogen. © 1994 Wiley-Liss, Inc.