Semi-solid Phase Research Articles

High-resolution insertion-site analysis by linear amplification–mediated PCR (LAM-PCR)

Integrating vector systems used in clinical gene therapy have proven their therapeutic potential in the long-term correction of immunodeficiencies. The integration loci of such vectors in the cellular genome represent a molecular marker unique for each transduced cell and its clonal progeny. To gain insight into the physiology of gene-modified hematopoietic repopulation and vector-related influences on clonal contributions, we have previously introduced a technology--linear amplification-mediated (LAM) PCR--for detecting and sequencing unknown DNA flanking sequences down to the single cell level (Supplementary Note online). LAM-PCR analyses have enabled qualitative and quantitative measurements of the clonal kinetics of hematopoietic regeneration in gene transfer studies, and uncovered the clonal derivation of non-leukemogenic and leukemogenic insertional side effects in preclinical and clinical gene therapy studies. The reliability and robustness of this method results from the initial preamplification of the vector-genome junctions preceding nontarget DNA removal via magnetic selection. Subsequent steps are carried out on a semisolid streptavidin phase, including synthesis of double complementary strands, restriction digest, ligation of a linker cassette onto the genomic end of the fragment and exponential PCR(s) with vector- and linker cassette-specific primers. LAM-PCR can be adjusted to all unknown DNA sequences adjacent to a known DNA sequence. Here we describe the use of LAM-PCR analyses to identify 5' long terminal repeat (LTR) retroviral vector adjacent genomic sequences.

On the solidification microstructure of Mg–30Zn–2.5Y metal–intermetallic alloy

The solidification microstructures of the Mg–30Zn–2.5Y alloy have been studied in detail using both optical and analytical electron microscopy. CALPHAD simulation has been carried out for in-depth understanding of the solidification process. The work clarifies phase equilibrium in the vicinity of the Mg corner of the Mg–Zn–Y ternary system, showing that a ternary icosahedral (I) quasicrystal phase is in equilibrium with the (Mg) solid solution phase. The suppression of the Z-phase during solidification is attributed to low thermodynamic driving force and sluggish transformation kinetics for ordering icosahedral clusters into the crystalline structure. The current work lays the foundation for developing (Mg)–matrix composites with a ternary icosahedral phase as well as other intermetallic compounds. The work presents a case study of solidification process to demonstrate the feasibility to cast (Mg)–matrix composite containing the I-phase from the L + I semi-solid phase region, as long as the equilibrium volume fraction of the I-phase is not too high to guarantee practical slurry fluidity before casting. This would be useful for making use of primary I-phase particles as effective nucleation sites to improve structural refinement and thus maximize the benefit from the I-phase reinforcement.

Continuous solidification of YBa 2Cu 3O 7− x by isothermal undercooling

Growth kinetics and microstructural development of single crystal YBa 2Cu 3O 7− x (Y123) superconductors, prepared by a melt texturing method under isothermal undercooling conditions, were investigated. At small undercooling, Δ T = 6 K, the initial growth of Y123 crystal was terminated at a size of ∼8 mm × 8 mm in the semisolid phase before the entire sample was fully solidified through the peritectic reaction. With incremental increase of Δ T, the growth of the crystal continued so that the whole sample (22 mm diameter and 8 mm thickness) solidified as a single crystal. Termination of the crystal growth at a given undercooling temperature was attributed to the coarsening process of Y211 particles in the melt associated with their surface energy contributions. Isothermal growth of large size Y123 single crystal requires a continuous supply of Y solute to the solidification interface which can be achieved by continuing dissolution of coarsening Y211 particles through further undercooling of the melt.

Codevelopment of dendritic cells along with erythroid differentiation from human CD34 + cells by tumor necrosis factor-α

Objective Tumor necrosis factor-α (TNF-α) inhibits erythropoiesis and enhances nonerythroid colony formation. The present study examines the nature of these nonerythroid cells and investigates their physiologic role in relation to erythroid progenitor cells. Materials and Methods Highly purified human CD34 + cells underwent erythroid differentiation in the presence of multiple cytokines, including stem cell factor (SCF), interleukin-3 (IL-3), and erythropoietin (EPO), with and without TNF-α. We enumerate colony-forming unit-erythroid (CFU-E) and glycophorin A (GPA; a specific marker for erythroid lineage) positive cells in semisolid phase as well as in liquid suspension culture. The character and roles of codeveloping nonerythroid cells in the presence of TNF-α were analyzed using fluorescent activating cell sorter, enzyme immunohistochemistry, and confocal microscopy. Results TNF-α inhibited the generation of GPA + cells and conversely enhanced the generation of GPA − cells. The GPA − cells were comprised of cells with excentric cell shape and were positive for HLA class I, HLA class II, CD1a, CD4, CD11c, CD14, CD40, CD80, CD83, and CD86, but not for CD3, CD8, CD19, CD20, and CD56, indicating the codevelopment of dendritic cells (DC) along with erythroid differentiation. Developing DC/DC precursors were detected within 3 days of culture. Only in the presence of TNF-α did CD34 + cells proliferate by forming aggregates where both GPA + and CD11c + DC/DC precursors were present. During culture period, immature CD11c + DC were capable of endocytosing damaged GPA + cells. Conclusions GPA − cells cogenerated from human CD34 + cells during erythroid differentiation in the presence of IL-3/SCF/EPO and TNF-α express DC phenotypes. The CD11c + DC subset physically and selectively associates with developing immature erythroid cells and damaged self-GPA + cells and then obtains and captures self-substances.

COMPARATIVE USE OF A COMMERCIAL BIOREACTOR SYSTEM AND CONVENTIONAL MICROPROPAGATION FOR THE PRODUCTION OF POTATO MICROTUBERS AND GRAPE MYRTLE (LAGERSTROEMIA INDICA) MICROSHOOTS

Potato minitubers and Lagerstroemia indica (grape myrtle) microshoots produced in vitro by a bioreactor system by Osmotek Ltd. (Israel) were compared by qualitative and qualitative means to respective ones produced conventionally in glass culture jars. 'Spunta' potato single node segments were cultured for 12 weeks, in complete darkness, in both liquid (LifeReactor©) and semi-solid phase (500 ml glass jars). The composition of media used was a) MS + 1.3 mg l -1 alar (daminozide) and b) CR (Charles and Rosssignol, 1993) + MS micro-salts and vitamins + 0.16 mg l -1 alar. Sucrose concentration was 120 g l -1 . The number of microtubers produced in the bioreactor was two times higher than produced in the jars but they were malformed, resulting in lower sprouting rates (3%) than the ones produced in agar solidified medium (41%). Lagerstroemia indica explants consisting of three nodes were cultured for 4 weeks in WPM + 1 μM BA + 0.3 μM NAA in liquid (LifeReactor©) and semi solid media exactly as described above under 40 μmol m 2 sec -1 of fluorescent light. No difference between numbers of plantlets produced by the two procedures was noticed, although the ones derived from liquid media were extensively hyperhydrized. Respective percentages of survival rates were 96.1% and 23.5% after acclimatization for plants from cultures on semi solid media compared to those out of the bioreactor.

Spreading and solidification of a molten microdrop in the solder jet bumping process

This work develops a model to predict the spreading and solidification of solder droplets deposited on a solid pad in the solder jet bumping process. The variational principle is employed to solve the fluid flow and the semi-solid phase is modeled as a non-Newtonian slurry. This modeling greatly saves the computational expenses of conventional numerical procedures. The simulations reveal that the substrate temperature is the single dominant controlling parameter that determines the final bump diameter (or height) when the substrate possesses a high effusivity. When the effusivity of a substrate is relatively low, both the substrate temperature and the droplet temperature at impact play important roles in determining the final bump diameter. Our model can be used in designing the experimental conditions to find the optimal process conditions for a desired bump geometry.

A simple semisolid subtraction method using carbodiimide-coated microplates.

In this article, we develop a novel subtraction method using carbodiimide-bound microplates. This method utilizes the high affinity of carbodiimides for both single- and double-stranded nucleic acids. Carbodiimide-mediated end-attachment of driver RNA to microplates allows semisolid phase hybridization between driver RNA and target cDNA, and ensures easy removal of RNA/cDNA hybrids composed of the genes commonly expressed in driver and target. As a result, the target-specific genes are left unhybridized and enriched in the hybridization supernatant. We define the optimal conditions for the method as a target/driver RNA ratio of 1:10 and a period of hybridization of 24 h. There are at least three major advantages with the present method: (1) The entire procedure, which consists of two steps, is very simple; (2) hybridization efficiency can be monitored before further processing of the samples; and (3) rare transcripts can be effectively enriched. This method may be a powerful tool to isolate the genes specifically expressed in particular cell or tissue types, and is easily applicable to many studies in molecular biology and genetics. Isolation of polyploid megakaryocyte-specific genes is shown as an example.

Partitioning invertase between a dilute water solution and generated droplets.

Water droplets or mist occur naturally in the air at seashores. These water droplets carry inorganic and organic substances from the sea to the land via the air, creating fertile land in sandy coastal areas (1). The same phenomenon occurs in an air-fluidized bed bioreactor (2). In an air-fluidized bed reactor, proteins can be transferred from the bioreactor semisolid bulk phase to an enriched droplet phase. This protein transfer process (droplet fractionation) can be experimentally simulated by shaking a separatory funnel containing a dilute solution of a given protein, which can be an enzyme like invertase. The created droplets become richer in invertase (protein) than that of the original dilute solution. The droplets can then be coalesced by trapping them and recovering the concentrated protein in the new liquid phase. Typically, in such a droplet fractionation process a collected enzyme can be degraded in its ability to catalyze a chemical reaction. In this article, we explore whether the initial solution pH control variable can be adjusted to minimize the decrease of enzyme activity in this process. The protein droplet recovery problem is one in which the recovered amount of desired protein (enzyme) in the droplet is maximized, subject to the minimization of the enzyme activity loss. The partition coefficient, which is the ratio between the protein concentration in the droplets and the residual solution, is maximized at approx 4.8 and occurs at pH 3.0. Here, the partition coefficient for invertase decreases as the initial solution pH increases, between pH 3.0 and 8.0. Interestingly, the initial solution surface tension seems to be inversely proportional to the partition coefficient. The partition coefficient reaches a maximum value at a surface tension value of approx 63 mN/m at pH 3.0. The enzymatic activity of the initial, the residual, and the droplet solutions all decrease as the bulk solution pH increases. A decrease of enzymatic activity was observed in the residual bulk solution when compared with that in the initial bulk solution at all pH levels. Also, up to 90% of the invertase activity was lost in the droplets when compared to the initial bulk solution.

Luminescence Properties of Salicylate Doped Zinc Benzoate

The salicylate-doped zinc benzoates Zn(Bzo)2: Sal are synthesized by means of the semisolid phase reaction method. The excitation and emission spectra are investigated, and compared with Zn(Sal)2, A-Zn(o-OC6H4CO2) and B-Zn(o-OC6H4Co2). For Zn(Bzo)2:Sal0.05, the luminescent intensity is mainly dependent on the efficiency of n,φ2 transition emission, the peaks of excitation and emission bands are at 328 and 384 nm, respectively. The emission intensity of Zn(Bzo)2: Sal0.05 is 3 times as strong as that of Zn(Sal)2, 1.6 times A-Zn(o-OC6H4CO2), and 6 times B-Zn(o-OC6H4CO2).

Innovative Design and Material Solutions of Thermal Contact Layers for High Heat Flux Applications in Fusion Devices

One difficulty associated with the design and development of sacrificial plasma facing components that have to handle the high heat and particle fluxes in ITER is achieving the necessary contact conductance between the plasma protection material and the high-conductivity substrate in contact with the coolant. This paper presents a novel bond idea which is proposed as one of the options for the sacrificial energy dump targets located at the bottom of the divertor legs. The bonded joint in this design concept provides thermal and electrical contact between the armour and the cooled sub-structure while promoting remote, in-situ maintenance repair and an easy replaceability of the armour part without disturbing the cooling pipes or rewelding neutron irradiated materials. To provide reliable and demountable adhesion, the bond consists of a metal alloy, treated in the semi-solid phase so that it leads to a fine dispersion of a globular solid phase into a liquid matrix (rheocast process). This thermal bond layer would normally operate in the solid state but could be brought reversibly to the semi-solid state during the armour replacement simply by heating it slightly above its solidus temperature. Material and design options are discussed in this paper. Possible methods of installationmore » and removal are described, and lifetime considerations are addressed. In order to validate this concept within the ITER time-frame, a R&D programme must be rapidly implemented.« less

メルトドラッグ法におけるＡｌ―Ｓｉ合金薄板自由凝固面の半凝固成形

To produce sound free solidification surface of the strip, melt drag process, which is one of single roll processes, was improved. An additional roll (forming roll) was attached to the outer side of the nozzle of the original melt drag process. In this work, forming of the free solidification surface of the strip was applied at the semi-solid phase by the forming roll. Free solidification surface can be formed in the smooth and flat shape, and thickness distribution of the cross section becomes uniform. The strip with 0.5 to 1.5mm thickness can be cast. Roll speed of this process is as fast as that of the original melt drag process (20-70m/min). The semi-solid region is formed by a roll and the forming load is very low, thus, the strip does not stick to the roll. Formability of the free solidification surface depends on the flow stress at the semi-solid phase.

In Situ-Forming Gels for Ophthalmic Drug Delivery

Poor bioavailability of ophthalmic solutions caused by dilution and drainage from the eye can be overcome by using in situ-forming ophthalmic drug delivery systems prepared from polymers that exhibit reversible phase transitions. Joshi et al. (1), have demonstrated that aqueous compositions that reversibly gel in response to simultaneous variations in at least two physical parameters, such as temperature, pH, and ionic strength, can be formed by appropriate combinations of macromolecular polymers which exhibit reversible gelation properties. In the present study, the rheological characterization of such a system, prepared by a combination of Carbopol (C) and methyl cellulose (MC), was carried out at two different pH (4.0 and 7.4) and temperatures (25 and 37 degrees C) by rotational cone and plate viscometry. The shear stress (tau) vs. shear rate (D) flow curves of the aqueous polymer solutions indicated a pseudoplastic behavior, with a yield point. An increase in pH from 4.0 to 7.4, or temperature from 25 to 37 degrees C, resulted in an increase in viscosity (eta), tau, and yield point, the magnitude of changes being highest when both the parameters were altered simultaneously. An increase in concentration of either C or MC, or an increase in MC molecular weight results in an increase in eta, tau, and yield point. Among the compositions studied, a solution containing 1.5% MC 0.3% C was found to have low eta, and formed a strong gel under simulated physiological conditions. Such a system can be formulated as drug containing liquid suitable for administration by instillation into the eye, which upon exposure to physiological conditions will shift to the gel (semi-solid) phase, thus increasing the precorneal residence time of the delivery system and enhancing ocular bioavailability.

C-kit Ligand Reactivates Fetal Hemoglobin Synthesis in Serum-Free Culture of Stringently Purified Normal Adult Burst-Forming Unit-Erythroid

AbstractWe have analyzed the reactivation of fetal hemoglobin (HbF) synthesis under rigorous in vitro conditions, ie, in mature erythroblasts generated by erythroid burst-forming units (BFU-E) stringently purified from normal adult peripheral blood and grown in fetal calf serum (FCS)-free semisolid or liquid phase culture. In clonogenetic dishes, graded amounts of c-kit ligand (KL) were added together with saturating levels of erythropoietin (Ep) and variable amounts of interleukin-3 and granulocyte-macrophage colony stimulating factor (IL-3/GM-CSF), ie, high or low level, or no IL-3/GM-CSF addition. In all conditions, KL induced a sharp, dose-dependent increase in the percentage of F cells and HbF content from nearly normal levels (<10% and <2.5%, respectively, at 0.1 and 1 ng/mL) up to 40% to 50% and 10% to 15% at 100 to 200 ng/mL. This increase was not associated with significant differences of burst number or stage of maturation at the time of analysis (as evaluated on the basis of percent mature erythroblasts and Hb content per cell). However, the KL-induced reactivation of HbF synthesis was strictly and directly correlated with a sharp increase of colony size, ie, cell number per burst. Addition of large amounts of IL-3 and GM-CSF (10 to 100 U and 1 to 10 ng/mL, respectively) significantly potentiated the KL-induced reactivation of HbF, as compared with low levels (0.1 U and 0.01 to 0.1 ng) or no addition of these growth factors: this increase was highly significant at low KL doses (ie, 1 to 10 ng/mL). Single-burst analysis showed that the KL-induced HbF reactivation occurs homogeneously in the erythroid colonies within each of these culture conditions. We have analyzed the effect of KL in liquid phase BFU-E culture treated with the IL-3/GM-CSF/Ep combination at sequential times until terminal erythroid maturation: KL causes a sharp increase in the percentage of F cells and HbF content in all stages of maturation, whereas the IL-3/GM-CSF/Ep combination alone has a markedly lower effect. These results suggest that KL plays a key role in the reactivation of HbF synthesis in adult life, whereas IL-3/GM-CSF potentiate this effect at low KL levels. The KL-induced HbF reactivation is seemingly related to an enhanced proliferation of erythroid progenitors in the erythropoietic differentiation pathway.

C-kit ligand reactivates fetal hemoglobin synthesis in serum-free culture of stringently purified normal adult burst-forming unit- erythroid

We have analyzed the reactivation of fetal hemoglobin (HbF) synthesis under rigorous in vitro conditions, ie, in mature erythroblasts generated by erythroid burst-forming units (BFU-E) stringently purified from normal adult peripheral blood and grown in fetal calf serum(FCS)- free semisolid or liquid phase culture. In clonogenetic dishes, graded amounts of c-kit ligand (KL) were added together with saturating levels of erythropoietin (Ep) and variable amounts of interleukin-3 and granulocyte-macrophage colony stimulating factor (IL-3/GM-CSF), ie, high or low level, or no IL-3/GM-CSF addition. In all conditions, KL induced a sharp, dose-dependent increase in the percentage of F cells and HbF content from nearly normal levels (&lt; 10% and &lt; 2.5%, respectively, at 0.1 and 1 ng/mL) up to 40% to 50% and 10% to 15% at 100 to 200 ng/mL. This increase was not associated with significant differences of burst number or stage of maturation at the time of analysis (as evaluated on the basis of percent mature erythroblasts and Hb content per cell). However, the KL-induced reactivation of HbF synthesis was strictly and directly correlated with a sharp increase of colony size, ie, cell number per burst. Addition of large amounts of IL- 3 and GM-CSF (10 to 100 U and 1 to 10 ng/mL, respectively) significantly potentiated the KL-induced reactivation of HbF, as compared with low levels (0.1 U and 0.01 to 0.1 ng) or no addition of these growth factors: this increase was highly significant at low KL doses (ie, 1 to 10 ng/mL). Single-burst analysis showed that the KL- induced HbF reactivation occurs homogeneously in the erythroid colonies within each of these culture conditions. We have analyzed the effect of KL in liquid phase BFU-E culture treated with the IL-3/GM-CSF/Ep combination at sequential times until terminal erythroid maturation: KL causes a sharp increase in the percentage of F cells and HbF content in all stages of maturation, whereas the IL-3/GM-CSF/Ep combination alone has a markedly lower effect. These results suggest that KL plays a key role in the reactivation of HbF synthesis in adult life, whereas IL- 3/GM-CSF potentiate this effect at low KL levels. The KL-induced HbF reactivation is seemingly related to an enhanced proliferation of erythroid progenitors in the erythropoietic differentiation pathway.

Thermophilic semisolid anaerobic digestion of municipal refuses

Comparison of both mesophilic (35 degrees C) and thermophilic (55 degrees C) anaerobic digestions of the organic fraction of municipal refuses in pilot digesters designed to process in a semisolid phase at total solids concentrations of ca. 25% shows that the average gas production is 20-25% higher in thermophilic conditions than in mesophilic conditions even for a retention time of 10 days. These results and the data recorded during long periods of experimentation indicate that the process allows to increase the net energy production and to improve the economical balance of an industrial plant.

Pharmacologic and biochemical modulation of human T-lymphocyte colony formation: Hormonal influences

The generation of T-cell colonies from human peripheral blood lymphocytes is a sensitive in vitro measure of cell-mediated immunity, considered to be under different and/or additional regulatory controls than short-term liquid cultures. The influences of steroids (aldosterone, estradiol, diethylstilbesterol, hydrocortisone, prednisolone, progesterone, testosterone), prostaglandins (PGA1, PGA2, PGB1, PGB2, PGE1, PGE2, PGF1α), bradykinin, cyclic adenosine monophosphate (AMP), cyclic guanosine monophosphate (GMP), epinephrine, glucagon, histamine, insulin, luteninizing hormone, luteotropic hormone, serotonin, and thyroxin on the generation of both T-cell colonies in semisolid phase and induction of transformation in liquid culture was assessed in parallel assays. Steroids uniformly suppressed both types of culture systems, although colony formation appeared more sensitive by several hours of magnitude. In contrast, significant differences in the response of lymphocytes in colony formation assay, compared to liquid transformation, was noted for the other agents. Prostaglandins significantly inhibited colony formation even in the presence of as little as 10−12 M PGE2; however, liquid culture responses were suppressed only by higher concentrations (10−15 M) and enhanced transformation was found at lower concentrations (10−9 M). Bradykinin, glucagon, and luteninizing hormone did not significantly influence either colony formation or liquid transformation. In contrast, cyclic AMP inhibited and cyclic GMP stimulated colony formation and liquid transformation. Histamine, insulin, epinephrine, and serotonin all had significant positive or negative influences on colony growth formation in concentrations that produced no detectable effects using conventional liquid transformation assays. Finally, correlation analysis of drug effects for each system extends the thesis that these assays quantitate different parameters of T-cell function. T-lymphocyte colony formation is a promising diagnostic tool for rapid screening of immune modulating agents.

Study of congenitally immunologic mutant New Zealand mice. V. B cell function of NZB-Xid mice.

NZB mice bearing the CBA/N X chromosome linked defect were generated by repetitive backcrossing and selection of the Xid gene. The male offspring resulting from the cross of NZB with CBA/N were selected as being XidY on the basis of sera IgM and IgG3 levels and responsiveness to DNP-Lys-Ficoll. Following this inbreeding protocol, 6th generation backcross NZB XidY mice were compared to littermate controls with respect to B cell function. Sera immunoglobulin levels of IgG1, IgG2b and IgA were similar in XidY and XY mice. In contrast, levels of IgM and IgG3, from XidY mice were approximately 15% and 50%, respectively, of values found in littermates. Furthermore, XidY mice failed to respond to DNP-Lys-Ficoll and had less than 3% splenic Lyb 5.1-bearing cells. Splenic immunoglobulin cell surface profiles, obtained by the fluorescent activated cell sorter, indicated a significant reduction in the frequency of Ig bearing cells in Xid animals. Such profiles were similar to those obtained for spleen cells from reference control CBA/N mice. Finally, an elevated number of splenic, lymph node and bone marrow background and lipopolysaccharide-induced B cell clones in semi-solid phase agar was found in NZB but not C57BL/6, C3H, BALB/c and DBA/2 controls. In contrast, NZB XidY mice had virtually no detectable B cell colonies. This data, obtained on significantly inbred XidY NZB mice, suggests that the Xid gene is dominant over several aspects of polyclonal B cell activation in NZB mice and indicates that serial observation of these mice will be valuable in understanding the interactions of genetic immunologic mutations and cellular function in autoimmunity.

Percutaneous Absorption of Steroids

where = concentration difference across the stratum corneum 2 stratum corneum thickness. Des = diffusion constant for the particular solute in the stratum corneum. Equation (1) is an expanded form of Fick's law which describes reasonably well the permeability of the skin to non-electrolytes. 4. These conclusions taken together with other results show that diffusion through the stratum corneum occurs with considerable difficulty even for small, soluble molecules (1). Steady state penetration through the stratum corneum is not primarily intercellular or intraappendageal. The hydrated stratum corneum seems best described as a dense, semi-solid phase into which small-molecular weight non-electrolytes dissolve with strong chemical interaction and through which diffusion occurs remarkably slowly. 5. In the application of equation 1 only the thickness, diffusivity and partition coefficient appropriate to the solute-solvent-membrane system have been considered. This simphfied picture is essentially correct, but applies only to steody stoic permeability. Before steady state diffusion is established (for) small molecules ordinarily within the first hour after the application of an aqueous solution) small but possibly significant amounts of material may diffuse more rapidly through hair follicles and sweat ducts. The reasons for this seemingly anomalous behavior arise from the limited area of the skin surface occupied by these appendages, their relatively large diffusiou constants and the non-linear character of diffusion prior to the steady state.