Topological defect coarsening in quenched smectic-C films analyzed using artificial neural networks.

Mechanically quenching a thin film of smectic-C liquid crystal results in the formation of a dense array of thousands of topological defects in the director field. The subsequent rapid coarsening of the film texture by the mutual annihilation of defects of opposite sign has been captured using high-speed, polarized light video microscopy. The temporal evolution of the texture has been characterized using an object-detection convolutional neural network to determine the defect locations, and a binary classification network customized to evaluate the brush orientation dynamics around the defects in order to determine their topological signs. At early times following the quench, inherent limits on the spatial resolution result in undercounting of the defects and deviations from expected behavior. At intermediate to late times, the observed annihilation dynamics scale in agreement with theoretical predictions and simulations of the 2D XY model.

Long- and short-term effects of biological hydrogels on capsule microvascular density around implants in rats.

Fibrous capsule formation around implants can inhibit solute exchange between implantable devices and the circulation. Parylene-n coated polycarbonate disks surrounded with growth factor reduced Matrigel (MG) or several gelatin-based matrices were implanted intramuscularly into rats for 21 or 50 days. MG supplemented with vascular endothelial growth factor (VEGF) or basic fibroblast growth factor (bFGF) increased capsule microvascular density at 21 days (p < 0.05) when compared to bare parylene-coated polycarbonate disks (control). The increased microvascular density around VEGF- and bFGF-treated implants regressed by 50 days and was no longer significantly different from controls. The microvascular density induced by the gelatin-based matrices was not significantly different from controls at 21 days, but was increased at 50 days (p < 0.05), suggesting a slower, long-term effect. Disks treated with MG and gelatin-based matrices had thinner capsules at 21 days (p < 0.05). By 50 days, the capsule thicknesses around these implants were no longer statistically thinner than controls. The capsule thickness around implants treated with VEGF, bFGF, and essential gelatin-based matrix was thinner than controls at 50 days (p < 0.05). These results indicate that it is possible to increase functional microvascular density within fibrous capsules using angiogenic growth factors and gelatin-based matrices. However, this effect may be short-lived, requiring chronic administration of growth factors.

Genes or gestation? Attitudes of women and men about biologic ties to children.

In light of the different reproductive roles of men and women, our objective was to ascertain whether men and women attach different significance to genetic, as distinct from gestational, ties to offspring. Surveys returned by 106 men and women of reproductive age in the general clinic waiting area of the University of Chicago Health Service showed that 71.1% of women and 62.9% of men would seek medical assistance if necessary to have a biologically related child. When women were asked whether they would choose the genetic tie or the gestational tie if both were not possible, 48.6% chose the genetic tie, and 51.4% chose the gestational tie. When men were asked which relationship they preferred for their partner, 73.5% chose the genetic tie, and 26.5% chose the gestational tie. Neither marital status nor experience of child-birth (for oneself or one's partner) were predictive of preferences. Our data suggest that gender differences as well as individual differences need to be addressed in counseling individuals and couples.

Relative stabilities of DNA three-way, four-way and five-way junctions (multi-helix junction loops): unpaired nucleotides can be stabilizing or destabilizing.

Competition binding and UV melting studies of a DNA model system consisting of three, four or five mutually complementary oligonucleotides demonstrate that unpaired bases at the branch point stabilize three- and five-way junction loops but destabilize four-way junctions. The inclusion of unpaired nucleotides permits the assembly of five-way DNA junction complexes (5WJ) having as few as seven basepairs per arm from five mutually complementary oligonucleotides. Previous work showed that 5WJ, having eight basepairs per arm but lacking unpaired bases, could not be assembled [Wang, Y.L., Mueller, J.E., Kemper, B. and Seeman, N.C. (1991) Biochemistry, 30, 5667-5674]. Competition binding experiments demonstrate that four-way junctions (4WJ) are more stable than three-way junctions (3WJ), when no unpaired bases are included at the branch point, but less stable when unpaired bases are present at the junction. 5WJ complexes are in all cases less stable than 4WJ or 3WJ complexes. UV melting curves confirm the relative stabilities of these junctions. These results provide qualitative guidelines for improving the way in which multi-helix junction loops are handled in secondary structure prediction programs, especially for single-stranded nucleic acids having primary sequences that can form alternative structures comprising different types of junctions.

Multiple dystrophin isoforms are associated with the postsynaptic membrane of Torpedo electric organ.

We have shown previously that dystrophin is a component of postsynaptic membranes in Torpedo electric organ and is localized at mammalian neuromuscular synapses. In skeletal muscle, dystrophin is also detectable at the non-synaptic membrane of the myofiber, whereas in the electric organ, dystrophin is strictly localized to the postsynaptic membrane, and is not detectable in non-synaptic membranes. Multiple isoforms of dystrophin are present in skeletal muscle, and different isoforms could potentially be targetted to synaptic and non-synaptic membranes. We sought to determine whether the electric organ contains a single, or multiple isoforms of dystrophin, and we show here that the electric organ contains both a and b isoforms of dystrophin. Because dystrophin is found only at the postsynaptic membrane of the electric organ, we conclude that the two isoforms coexist in the postsynaptic membrane.

Switches in macromolecular synthesis during induction of competence for transformation of Streptococcus sanguis.

The induction of synchronous development of competence for genetic transformation in Streptococcus sanguis, by either endogenous or exogenous competence factor (CF), is manifested in the transient synthesis of a new set of at least 10 polypeptides, ranging from 14,000 to 51,000 in molecular weight. Eight polypeptides (E14, E16, E24, E28, E32, E37, E44, E51) appear early, and two polypeptides (L34, L42) appear 5-10 min later. One of the newly synthesized early polypeptides, E16, is shown to be a component of the presynaptic complex containing single-stranded DNA that is produced in vivo upon uptake of native donor DNA. Concomitant with this induced synthesis of competence-specific polypeptides there is a net decrease in RNA and protein synthesis but no change in DNA synthesis; donor DNA-binding ability and transformability reach maxima during the phase of diminishing macromolecular synthesis. Subsequently, donor DNA-binding ability and transformability decay at disproportionate rates as cells return to the normal state of macromolecular synthesis within one generation. Coincident with the induction of competence, the synthesis of a new RNA transcript of high molecular weight appears to be induced which continues during the restricted phase of total cellular RNA synthesis.

Superhelical DNA in Streptococcus sanguis: role in recombination in vivo.

Competent Streptococcus sanguis treated with non-lethal doses of coumermycin A1 immediately before or after uptake of radioactive transforming DNA were reduced in their capacity to yield transformants. This treatment did not alter bacterial ability to bind DNA in DNase I-resistant form, nor did it prevent the single-stranded donor DNA-recipient protein complexes formed upon uptake at the surface of the bacteria from translocating to chromosomal sites. Inhibition of transformation by heterospecific DNA was greater than that by homospecific DNA. The reduction in transformant yield was not accompanied by any loss of donor counts incorporated into the recipient chromosome, but rather by a loss of genetic activity of incorporated donor material indicating a failure of genetic integration and degradation of donor DNA as a consequence of coumermycin treatment. The inhibitory effect of coumermycin on transformation was associated with in vivo loss of chromosomal DNA superhelicity, The chromosomal DNA remained intact, however, indicative of inhibition of a gyrase-like enzyme responsible for the maintenance of negative supercoiling of the S. sanguis chromosome. Upon treatment with the drug, a coumermycin-resistant mutant strain showed neither loss of chromosomal superhelicity nor any inhibitory effect on genetic integration of donor DNA. The evidence supports the idea that chromosomal superhelicity promotes genetic recombination in vivo.

Enhanced transformability with heterospecific deoxyribonucleic acid upon removal of nascent ribonucleic acid from the Streptococcus sanguis genome.

Treatment of Streptococcus sanguis recipient cells with rifampin (RIF) at the time of deoxyribonucleic acid (DNA) addition was an effective means of reducing discrimination, that is, of causing an increase in the number of transformants induced by irreversibly bound heterospecific DNA without significantly changing the number induced by bound homospecific DNA. RIF was unable to reduce discrimination when the recipient cells were RIF resistant due to an altered ribonucleic acid (RNA) polymerase. When recipient cells were treated at the time of DNA addition with concentrations of streptolydigin (STG) as inhibitory of RNA synthesis as RIF, discrimination was not reduced. The kinetics of RNA synthesis inhibition with these inhibitors indicated that, as reported for other bacterial species, RIF inhibited the initiation of transcription by RNA polymerase, whereas STG inhibited the progression of RNA polymerase at any point. Pulse-labeling of RNA immediately before STG addition showed that, if cells were incubated under STG inhibition for 10 to 15 min, their nascent RNA was degraded. Genome-bound RNA polymerase was not released under these conditions. When recipient cells were incubated with STG until nascent RNA was degraded and then exposed to transforming DNA, STG was as effective as RIF in reducing discrimination. The presence of nascent RNA was thereby implicated in the transforming inefficiency of incompletely homologous DNA.

Single-stranded regions in Streptococcus pneumoniae chromosomal deoxyribonucleic acid and their relation to transformation.

Deoxyribonucleic acid (DNA) in lysates of both completent and noncompetent streptococcus pneumoniae cells was characterized by chromatography on benzoylated, naphthoylated diethylaminoethyl-cellulose columns, by sensitivity to Aspergillus oryzae S1 endonuclease, and by sucrose gradient analysis. The DNAs from both competent and noncompetent cells were found to contain similar extents of single-stranded regions. These single-stranded regions appeared to be intact, unpaired regions in double-stranded DNA rather than gaps, nicks, or unpaired ends in the DNA. Inhibition of cells with rifampin prior to lysis increased the amount of such single strandedness in the DNA. Lysates made at various times after [14C]thymidine-labeled cells had bound [3H]thymidine-labeled transforming DNA were also characterized by benzoylated, naphthoylated diethylaminoethyl-cellulose chromatography. Changes in the elution profiles of DNA from cells exposed to homospecific (S. pneumoniae) donor DNA were indicative of the formation of complexes between donor DNA and the single-stranded regions of recipient DNA. In contrast, profiles of DNA from cells exposed to heterospecific (S. sanguis) DNA did not show significant changes, indicating that few such donor-recipient complexes were formed during heterospecific transformation.

Translocation of the pre-synaptic complex formed upon DNA uptake by Streptococcus sanguis and its inhibition by ethidium bromide.

Donor DNA in its initially bound, single-stranded form exists in a chromosomally-unassociated complex where it is resistant to exogenous DNase I but sensitive to micrococcal nuclease. Most of the complexes are readily recuperable from the supernatant of recipients converted into spheroplasts. Subsequent to formation of this superficially located complex, donor DNA progressively associates with the recipient chromosome into which it is eventually integrated. Treatment of recipients with ethidium bromide at various times after initial DNA binding almost immediately halts translocation of whatever donor material is not yet synapsed with the chromosome. On the other hand, donor DNA that has already synapsed experiences no difficulty in becoming genetically integrated. Some degradation occurs to DNA that fails to undergo translocation as a result of ethidium bromide treatment, the acid-soluble products appearing in the culture medium. DNA in untranslocated complexes surviving treatment is not appreciably different in single-strand length from that in untreated complexes. When these surviving complexes are isolated from a cell lysate, the contained DNA can be shown by spectrofluorometry to have bound the drug.

Specific inactivation of heterospecific transforming DNA by a factor derived from Streptococcus sanguis lysates.

A heat-sensitive factor obtained from lysates of competent Streptococcus sanguis cells reacts specifically with native DNA of heterospecific (S. pneumoniae or calf thymus) origin. In vitro it does not alter the double or single strand length of the DNA, nor does it affect uptake of the DNA by compentent S. pneumoniae cells in DNase I-resistant form. Following uptake, however, DNA previously exposed to the factor loses over 90% of its biological activity. Reaction of heterospecific DNA with the factor is competitive, suggesting a competition for binding to the factor. Heating treated DNA prior to its reaction with recipient cells, apparently by irreversibly dissociating the factor, restores to the DNA its original potential transforming activity. Specific activity of the factor can be increased in cells grown under certain conditions; this increase is blocked by erythromycin.

Fate of heterospecific transforming DNA bound to Streptococcus sanguis.

The fate of 3H-labeled str-r fus-s DNA from Streptococcus pneumoniae, bound after a 1-min uptake to 14C-labeled str-s fus-r S. sanguis recipients, was followed by techniques previously developed for analyzing the fate of homospecific DNA. Heterospecific S. pneumoniae DNA was bound and formed complexes with recipient protein in a manner similar to that of homospecific DNA but transformed relatively poorly. The rate at which complexed heterospecific DNA becomes physically associated with recipient DNA, and at which donor markers are integrated into the chromosome, was slower than in the case of homospecific DNA. In addition, about half of the heterospecific donor counts initially bound in trichloracetic acid-insoluble form were gradually solubilized and released from the cell. The association of heterospecific DNA with the recipient chromosome was more unstable than that involving homospecific DNA, since only associations of the former type were largely dissociated by isolation and resedimentation. The donor DNA-containing material so dissociated had the same sedimentation properties as complexed heterospecific DNA before association, indicating that the complex of single-stranded donor DNA and recipient protein formed on uptake moves as a whole from its site of formation to synapse with the chromosome.

Fate of homospecific transforming DNA bound to Streptococcus sanguis.

The fate of [3H]DNA from Streptococcus sanguis str-r43 fus-s donors in [14C]S. sanguis str-s fus-r1 recipients was studied by examining the lysates prepared from such recipients at various times after 1 min of exposure to DNA. The lysates were analyzed in CsCl and 10 to 30% sucrose gradients; fractions from the gradients were tested for biological activity and sensitivity to nucleases, subjected to various treatments and retested for nuclease sensitivity, and run on 5 to 20% neutral and alkaline sucrose gradients. The results demonstrate that donor DNA bound to S. sanguis cells in a form resistant to exogenous deoxyribonuclease is initially single stranded and complexed to recipient material. Donor DNA can be removed from the complex upon treatment of the complex with Pronase, phenol, or isoamyl alcohol-chloroform. Within the complex, donor DNA is relatively insensitive to S1 endonuclease but can regain its sensitivity by treatment with phenol. With time the complex moves as a whole to associate physically with the recipient chromosome. After a noncovalent stage of synapsis, donor material is covalently bonded to and acquires the nuclease sensitivity of recipient DNA, while donor markers regain transforming activity and become linked to resident markers.