Modeling electrokinetic flows with the discrete ion stochastic continuum overdamped solvent algorithm.

In this article we develop an algorithm for the efficient simulation of electrolytes in the presence of physical boundaries. In previous work the discrete ion stochastic continuum overdamped solvent (DISCOS) algorithm was derived for triply periodic domains, and was validated through ion-ion pair correlation functions and Debye-Hückel-Onsager theory for conductivity, including the Wien effect for strong electric fields. In extending this approach to include an accurate treatment of physical boundaries we must address several important issues. First, the modifications to the spreading and interpolation operators necessary to incorporate interactions of the ions with the boundary are described. Next we discuss the modifications to the electrostatic solver to handle the influence of charges near either a fixed potential or dielectric boundary. An additional short-ranged potential is also introduced to represent interaction of the ions with a solid wall. Finally, the dry diffusion term is modified to account for the reduced mobility of ions near a boundary, which introduces an additional stochastic drift correction. Several validation tests are presented confirming the correct equilibrium distribution of ions in a channel. Additionally, the methodology is demonstrated using electro-osmosis and induced-charge electro-osmosis, with comparison made to theory and other numerical methods. Notably, the DISCOS approach achieves greater accuracy than a continuum electrostatic simulation method. We also examine the effect of under-resolving hydrodynamic effects using a "dry diffusion" approach, and find that considerable computational speedup can be achieved with a negligible impact on accuracy.

Numerical simulation of a laboratory-scale turbulent V-flame.

We present a three-dimensional, time-dependent simulation of a laboratory-scale rod-stabilized premixed turbulent V-flame. The experimental parameters correspond to a turbulent Reynolds number, Re(t) = 40, and to a Damköhler number, D(a) = 6. The simulations are performed using an adaptive time-dependent low-Mach-number model with detailed chemical kinetics and a mixture model for differential species diffusion. The algorithm is based on a second-order projection formulation and does not require an explicit subgrid model for turbulence or turbulence/chemistry interaction. Adaptive mesh refinement is used to dynamically resolve the flame and turbulent structures. Here, we briefly discuss the numerical procedure and present detailed comparisons with experimental measurements showing that the computation is able to accurately capture the basic flame morphology and associated mean velocity field. Finally, we discuss key issues that arise in performing these types of simulations and the implications of these issues for using computation to form a bridge between turbulent flame experiments and basic combustion chemistry.

Herpes simplex encephalitis. A study of seven patients and their immunological response prior to routine acyclovir treatment.

OBJECTIVES: A retrospective study on cerebrospinal fluid (CSF) samples was made possible by access to a large CSF bank, which has been supplemented by the well characterised prior collection of CSF from patients with Herpes simplex encephalitis (including serial samples from some patients and kindly donated by Professor Maurice Longson, Manchester). These samples are of particular interest because they were collected prior to the routine administration of acyclovir. METHODS: Although an earlier study had shown that there was indeed a correlation between higher titres of antibody and a better outcome, the data did not emerge with statistical significance. The current study was based upon an improved method, which demonstrates the antigen-specific clonality of the immune response. RESULTS: The primitive polyclonal anamnestic response was contrasted with the strong antigen-specific response, which was manifested by a monoclonal pattern in some patients. A clear distinction emerged between two sub-groups of patients on the basis of these findings, which showed a statistically significant (P<0.03) correlation with outcome. CONCLUSIONS: This has allowed us to further support the hypothesis that a strong immunological response has positive prognostic value during the course of the disease.

The drosophila aeroplane mutant is caused by an I-element insertion into a tissue-specific teashirt enhancer motif.

In Drosophila melanogaster, aeroplane (ae) is a regulatory allele of teashirt (tsh), and the mutant wing posture phenotype of homozygous ae flies is caused by a defect in the hinge region of the wing, whereby the base of the wing at the proximal ventral radius is fused to the thorax in the region of the pleural wing process. The apparent paralysis of the wings and the drooping halteres are caused by an I-element insertion into a 3' noncoding sequence of tsh. The cis-acting regulatory element interrupted by the I element is required, to drive tsh expression in the regions of the developing adult that give rise to proximal wing and haltere tissues. Loss of this expression results in the fusion of the proximal structures of the wing and halteres to the thoracic cuticle. Further characterization of this tsh regulatory motif has now identified an additional enhancer activity directing tsh expression in tissues forming portions of the midgut. Subdivision of this midgut enhancer activity has identified putative negatively acting motifs.

Identification of a regulatory allele of teashirt (tsh) in Drosophila melanogaster that affects wing hinge development. An adult-specific tsh enhancer in Drosophila.

A cis-acting regulatory element defined herein is required to drive teashirt (tsh) expression in the regions of the developing adult that give rise to proximal wing and haltere tissues. Loss of this expression results in the fusion of the proximal structures of the wing and halteres to the thoracic cuticle. This represents the first description of a viable adult-specific regulatory allele of tsh with a visible phenotype, and it enlarges our understanding of the expression of tsh and its function during the development of the adult.

Rediscovery and further characterization of the aeroplane (ae) wing posture mutation in Drosophila melanogaster.

In 1931, Theodore Quelprud characterized a novel spontaneous mutation in Drosophila melanogaster, which was named aeroplane (ae) based on its abnormal wing posture. Although the characterization of the original ae locus was minimal, it is very likely that another allele of this extinct mutation has now been identified. aeroplane-like (ae-l) was isolated as a by-product of a transformation experiment. The apparent wing paralysis is not caused by any obvious abnormalities in the thorax, wing, indirect flight muscles or direct flight muscles. Classical genetic complementation analyses of ae-l with other genes in the region suggest that it represents an allele of a novel locus. Unexpectedly, a molecular examination revealed that the physical lesion identified in the ae-l mutant is exceptionally close to the homeotic gene teashirt (tsh) and, indeed, may represent an unusual allele of teashirt.

Molecular interactions between Vestigial and Scalloped promote wing formation in Drosophila.

Scalloped (Sd) and Vestigial (Vg) are each needed for Drosophila wing development. We show that Sd is required for Vg function and that altering their relative cellular levels inhibits wing formation. In vitro, Vg binds directly to both Sd and its human homolog, Transcription Enhancer Factor-1. The interaction domains map to a small region of Vg that is essential for Vg-mediated gene activation and to the carboxy-terminal half of Sd. Our observations indicate that Vg and Sd function coordinately to control the expression of genes required for wing development, which implies that Vg is a tissue-specific transcriptional intermediary factor of Sd.

The thermal explosion revisited.

The classical problem of the thermal explosion in a long cylindrical vessel is modified so that only a fraction alpha of its wall is ideally thermally conducting while the remaining fraction 1-alpha is thermally isolated. Partial isolation of the wall naturally reduces the critical radius of the vessel. Most interesting is the case when the structure of the boundary is a periodic one, so that the alternating conductive alpha and isolated 1-alpha parts of the boundary occupy together the segments 2pi/N (N is the number of segments) of the boundary. A numerical investigation is performed. It is shown that at small alpha and large N, the critical radius obeys a scaling law with the coefficients depending on N. For large N, the result is obtained that in the central core of the vessel the temperature distribution is axisymmetric. In the boundary layer near the wall having the thickness approximately 2pir0/N (r0 is the radius of the vessel), the temperature distribution varies sharply in the peripheral direction. The temperature distribution in the axisymmetric core at the critical value of the vessel radius is subcritical.

A genetic and molecular analysis of an invectedDominant mutation in Drosophila melanogaster.

The invected gene of Drosophila melanogaster is a homeobox-containing gene that is closely related to engrailed. A dominant gain of function allele, invectedDominant, was derived from mutagenesis of a dominant allele of vestigial, In(2R)vgW. A careful analysis of the phenotype of invectedDominant shows that it is associated with a transformation of the anterior compartment of the wing to a posterior fate. This transformation is normally limited to the wing blade itself and does not involve the remaining tissues derived from the wing imaginal disc, including the wing hinge and dorsal thorax of the fly. The ectopic expression of invected protein associated with invectedDominant correlates spatially with the normal expression pattern of vestigial in the wing imaginal disc, suggesting that control elements of vestigial are driving ectopic invected expression. This was confirmed by sequence analysis that shows that the dominant vestigial activity was eliminated by a deletion that removes the 3' portion of the vestigial coding region. This leaves a gene fusion wherein the vestigial enhancer elements are still juxtaposed immediately 5' to the invected transcriptional start site, but with the vg sequences harboring an additional lesion. Unlike recessive invected alleles, the invectedDominant allele produces an observable phenotype, and as such should prove useful in determining the role of invected in patterning the wing imaginal disc. Genetic analysis has shown that mutations of polyhomeotic, a gene involved in regulating engrailed expression, cause a reproducible alteration in the invectedDominant phenotype. Finally, the invectedDominant allele should prove valuable for identifying and characterizing genes that are activated within the posterior compartment. A screen using various lacZ lines that are asymmetrically expressed in an anterior-posterior manner in the wing imaginal disc isolated one line that shows posterior-specific expression within the transformed anterior compartment.

Base miscoding and strand misalignment errors by mutator Klenow polymerases with amino acid substitutions at tyrosine 766 in the O helix of the fingers subdomain.

A mutant derivative of Klenow fragment DNA polymerase containing serine substituted for tyrosine at residue 766 has been shown by kinetic analysis to have an increased misinsertion rate relative to wild-type Klenow fragment, but a decreased rate of extension from the resulting mispairs (Carroll, S. S., Cowart, M., and Benkovic, S. J. (1991) Biochemistry 30, 804-813). In the present study we use an M13mp2-based fidelity assay to study the error specificity of this mutator polymerase. Despite its compromised ability to extend mispairs, the Y766S polymerase and a Y766A mutant both have elevated base substitution error rates. The magnitude of the mutator effect is mispair-specific, from no effect for some mispairs to rates elevated by 60-fold for misincorporation of TMP opposite template G. The results with the Y766S mutant are remarkably consistent with the earlier kinetic analysis of misinsertion, demonstrating that either approach can be used to identify and characterize mutator polymerases. Both the Y766S and Y766A mutant polymerases are also frameshift mutators, having elevated rates for two-base deletions and a 276-base deletion between a direct repeat sequence. However, neither mutant polymerase has an increased error rate for single-base frameshifts in repetitive sequences. This error specificity suggests that the deletions generated by the mutator polymerases are initiated by misinsertion rather than by strand slippage. When considered with recent structure-function studies of other polymerases, the data indicate that the nucleotide misinsertion and strand-slippage mechanisms for polymerization infidelity are differentially affected by changes in distinct structural elements of DNA polymerases that share similar subdomain structures.

Role of CD34+ cells in engraftment after high-dose melphalan in multiple myeloma patients given peripheral blood stem cell rescue.

During the period December 1992 to June 1995, 95 patients were treated with high-dose melphalan (HDM) with peripheral blood stem cell rescue (PBSCR). Sixty-five had received previous treatment and 28 had relapsed. Among patients who had relapsed 21/28 had received HDM previously including one who received HDM twice during the course of the study. Seventy-five patients were given HDM/PBSCR for the first time. Comparisons have been made between engraftment times for platelets and neutrophils among patients who received less than or greater than 2 x 10(6) CD34+ cells at rescue. Analyses have also been done to evaluate the effect of previous HDM on recovery. Mobilization of progenitor cells was done with granulocyte colony-stimulating factor (G-CSF). Patients received only PBSCR. No growth factors were given to the PBSCR recipients during the recovery period. The percentage of patients from whom the number of CD34+ cells mobilized was > 2 x 10(6)/kg was similar in patients who received HDM for the first time (23%) compared with those who had had it previously (19%). The yield of CD34+ cells correlated with the number of granulocyte-macrophage colony forming units (CFU-GM). Although the number of CD34+ cells infused was < 2 x 10(6)/kg in 77% of patients, all engrafted for neutrophils to > 0.5 x 10(9)/l. This was delayed in patients who had had previous HDM (P < 0.02). Platelet recovery to > 25, 50 and 100 x 10(9)/l was delayed in all patients who received < 2 x 10(6) CD34+ cells/kg infused (P < 0.02). In patients who had had previous HDM both neutrophil (P < 0.05) and platelet recovery (P < 0.007) were delayed compared with recovery in patients who had not had HDM. In patients who had had previous HDM and received < 2 x 10(6) CD34+ cells/kg infused only 3/17 regained platelets to > 100 x 10(9)/l compared with 3/4 who had > 2 x 10(6) CD34+ cells/kg infused (P < 0.05 Fisher's exact test). There was no evidence that low numbers of CD34+ cells in the PBSCR were associated with early death. The data show that previous treatment with HDM had adverse effects on the subsequent engraftment of platelets among patients given HDM/PBSCR. The data suggest that additional measures are needed to achieve platelet reconstitution in these heavily pre-treated patients.

Lymphocyte recovery and clinical response in multiple myeloma patients receiving interferon alpha 2 beta after intensive therapy.

The recovery of immunoregulatory cells in the peripheral blood of patients with multiple myeloma receiving maintenance therapy with interferon alpha 2 beta (IFN-alpha 2 beta) after intensive therapy with high-dose melphalan and autologous bone marrow or peripheral blood stem cell rescue was studied. IFN-alpha 2 beta significantly inhibited the recovery of CD3+, CD4+, CD8+, CD56+/CD3- and CD16+/CD3- lymphocytes compared with numbers found in patients who had no further post-transplant treatment, but had no effect on the recovery of CD19+ cells. Among patients who did not receive IFN-alpha 2 beta, the number of CD8+, CD56+/CD3- and CD16+CD3- lymphocytes recovered to values similar to normal volunteers with increasing time after intensive therapy, however the number of CD4+ cells remained significantly below levels found in normal volunteers. Although CD16+/CD3- and CD56+/CD3- cell numbers were reduced in patients receiving IFN-alpha 2 beta, natural killer (NK) activity was not affected. The levels of soluble interleukin 2 receptor (sIL-2R) were similar in all patients and IL-2 was not detected in any patient. At the time of writing, of the total of 69 patients, seven have relapsed, of whom three were receiving IFN-alpha 2 beta, however there was no correlation between the absolute numbers of any lymphocyte subset with imminent relapse. The data suggest that the recovery of a specific lymphocyte subset(s) in peripheral blood is unlikely to be associated with the maintenance of response after intensive therapy.

2',5'-Oligoadenylate synthetase levels in patients with multiple myeloma receiving maintenance therapy with interferon alpha 2b do not correlate with clinical response.

In clinical trials with interferon alpha 2b (IFN-alpha 2b) as maintenance therapy for multiple myeloma, the therapeutic benefit is inconclusive. Although the mechanism(s) by which IFN-alpha 2b prolongs remission in some patients is unknown, 2',5'-oligoadenylate synthetase (2,5-A synthetase) has been used as an objective indicator that IFN-alpha 2b is active in vivo. The enzyme was assayed in cytosol preparations of peripheral blood mononuclear cells (MNCs) from 111 patients who were receiving IFN-alpha 2b and 54 patients who were not, using an assay which measures the conversion of [alpha-32P]ATP to triphospho(adenylyl 2',5')adenosine. 2,5-A synthetase activity was compared with response to intensive therapy and with duration of maintenance therapy. Seventy-three per cent of patients had measurable amounts of 2,5-A synthetase during the first 6 months of maintenance therapy. This percentage decreased with longer follow-up but not significantly. There was no difference between the magnitude of enzyme induction amongst patients who were in complete remission, partial response or who had no change in disease status following intensive therapy. Peripheral blood T cells were a major source of 2,5-A synthetase activity in patients receiving the cytokine. However, both T and B cells produced the enzyme following exposure to IFN-alpha in vitro. The data show that the level of 2,5-A synthetase in patients with multiple myeloma is not indicative of clinical response to IFN-alpha 2b.

Distinguishable functions for engrailed and invected in anterior-posterior patterning in the Drosophila wing.

Subdivision of the limb primordia into compartments initiates pattern formation in the developing limbs. Interaction between distinctly specific cells in adjacent compartments leads to localized expression of the secreted signalling molecules Wingless (Wg) or Decapentaplegic (Dpp), which in turn organize pattern and control growth of the limbs. The homeobox gene engrailed has been implicated in specification of posterior cell fate, whereas the LIM/homeobox gene, apterous, specifies dorsal fate. Removing apterous activity causes a complete transformation from dorsal to ventral fate and leads to the formation of an ectopic dorsal-ventral boundary organizer. By contrast, removing engrailed activity causes incomplete morphological transformation from posterior to anterior fate in the wing, and fails to produce an ectopic anterior-posterior organizer (reviewed in ref.2). Complete transformation can only be effected by simultaneously eliminating activity of engrailed and its homologue invected. Here we show that invected functions principally to specify posterior cell fate. Thus establishment of the anterior-posterior organizer and control of compartment identity are genetically distinguishable, and invected may perform a discrete subset of functions previously ascribed to engrailed.

G-CSF is a major component of colony-stimulating activity (CSA) in the plasma of patients with multiple myeloma after treatment with high-dose melphalan (HDM).

Colony-stimulating activity (CSA) was measured by the production of granulocyte-macrophage colony-forming units (GM-CFU) from normal donor bone marrow in the plasma of 29 patients with multiple myeloma (MM) after intensive treatment with high-dose melphalan (HDM) with or without autologous bone marrow rescue (ABMR). Although patients who received ABMR had an earlier recovery of circulating neutrophils compared with those who received HDM alone, the time at which CSA reached a maximum was similar in both groups (10 to 11 days) after therapy. The decline in CSA correlated with the recovery of the neutrophil count. In plasma from patients who received recombinant human granulocyte colony-stimulating factor (rhG-CSF), in addition to an autograft, CSA reached a maximum earlier (7 days). Furthermore, neutrophil recovery was earlier in these patients. Platelet recovery was not increased by rhG-CSF. The time at which CSA was maximum in four patients who were undergoing intensive therapy for the second time occurred 9 days after treatment with HDM. Although the period without neutrophils was longer in three (of four) patients who survived long term, one patient who received rhG-CSF had a shorter period of neutropenia than the two who had not had the cytokine. G-CSF was detected in plasma from seven of seven patients but not at all times after treatment. In plasma samples that contained G-CSF, colony numbers were increased by recombinant interleukin-4 (rIL-4) in vitro. Neither IL-3 nor GM-CSF was detected in plasma; however, antibody to GM-CSF reduced CSA in all samples after intensive therapy. The data suggest that CSA is a consistent physiologic response to intensive therapy, even in previously treated patients, but that hematologic recovery is dependent on the availability of viable progenitor cells.

Protected P-element termini suggest a role for inverted-repeat-binding protein in transposase-induced gap repair in Drosophila melanogaster.

P-element transposition is thought to occur by a cut-and paste mechanism that generates a double-strand break at the donor site, the repair of which can lead to internally deleted elements. We have generated a series of both phenotypically stronger and weaker allelic derivatives of vg21, a vestigial mutant caused by a P-element insertion in the 5' region of the gene. Virtually all of the new alleles arose by internal deletion of the parental element in vg21, and we have characterized a number of these internally deleted P elements. Depending upon the selection scheme used, we see a very different spectrum of amount and source of P-element sequences in the resultant derivatives. Strikingly, most of the breakpoints occur within the inverted-repeats such that the last 15-17 bp of the termini are retained. This sequence is known to bind the inverted-repeat-binding protein (IRBP). We propose that the IRBP may act to preserve the P-element ends when transposition produces a double-strand gap. This allows the terminus to serve as a template upon which DNA synthesis can act to repair the gap. Filler sequences found at the breakpoints of the internally deleted P elements resemble short stretches, often in tandem arrays, of these terminal sequences. The structure of the filler sequences suggests replication slippage may occur during the process of gap repair.

The proliferation of multiple myeloma colonies (MY-CFUc) in vitro is independent of prognosis and is not associated with mutated N- or K-ras alleles in human bone marrow aspirates.

During the period September 1987 to March 1993 the proliferation of myeloma cells as colonies (MY-CFUc) in vitro was examined in bone marrow aspirates from 43 patients with multiple myeloma and two patients with Waldenström's macroglobulinaemia. Twenty-four samples from 45 patients, of whom three were at presentation, four were in complete remission (CR), six had achieved a partial response (PR) and 11 had progressive disease (PD), produced MY-CFUc in vitro. The same bone marrow aspirates or one taken within 2 months of that assessed for MY-CFUc were used in the polymerase chain reaction (PCR). Genomic DNA was analysed for mutations in N- and K-ras by slot blotting of the amplified products from the PCR with 32P-labelled probes and by direct sequencing. No mutations were detected in N- or K-ras proto-oncogenes at codons 12, 13 or 61 in any sample. Eleven of the patients from whom MY-CFUc were produced remain alive with a median survival of 73 months (range 15-75 months). MY-CFUc have been cultured from 19 of these 24 patients on subsequent occasions, of whom nine remain alive. Among patients whose cells did not produce MY-CFUc in vitro at the time of sampling for mutated ras alleles, biopsy samples from four patients have produced MY-CFUc in vitro on subsequent occasions, of whom one patient remains alive. The data show that the proliferation of MY-CFUc in vitro occurred independently of disease status and was not indicative of prognosis. The failure to detect mutated N- or K-ras alleles in any sample suggests that if such mutations were present in the cells which form colonies in vitro they represented less than 0.1% of the tumour burden and did not affect the survival of this group of patients.

Neutralising antibodies in patients with multiple myeloma receiving maintenance therapy with interferon alpha 2b.

In a study of 29 patients who were receiving or had received interferon alpha 2b (IFN-alpha 2b) as maintenance therapy for multiple myeloma, antibodies were detected in 58% (17/29) of patients measured by a solid-phase enzyme-linked immunosorbent assay (ELISA). Only 7/17 patients who were positive for antibody in the ELISA had neutralising antibody to IFN-alpha 2b, measured by virus growth inhibition. These patients comprised six who were receiving IFN-alpha 2b at the time of assessment and one who had finished treatment. Among patients who were receiving the cytokine, four had progressive disease, one was in complete remission and one in partial remission. Neutralising activity was also detected to natural human leucocyte IFN-alpha in the same patients. Two patients who were positive for neutralising antibody remain in remission and are continuing to receive IFN-alpha 2b. These two patients have since lost their neutralising titre. No neutralising antibody to IFN-alpha 2b or natural human leucocyte IFN-alpha was detected in serum from six normal donors. The data suggest that neutralising antibody formation in patients with multiple myeloma is not responsible for relapse in patients receiving IFN-alpha 2b. The transient nature of neutralising antibody production in patients who remain in remission suggests that this response to IFN-alpha 2b is not associated with memory B cells.

Targeting of an enhancer trap to vestigial.

The vestigial gene (vg+) is required for normal wing development and is expressed in a spatially distinct pattern in imaginal discs. We have exploited a general property of P element alleles to target an enhancer trap to the 5' region of the gene. By replacing the P element resident at this site in vg21 with a P element carrying a lacZ reporter gene, the vglacZ1 allele was selected on the basis of its increased mutant phenotype. In contrast to vg+ expression, which occurs primarily in the presumptive wing margin and hinge, beta-galactosidase expression in vglacZ1 wing discs is localized to the dorsal wing surface and displays homologous haltere expression. The targeting of P element enhancer traps could be readily extended to other genes with low rates of primary P element insertion.