Phase-coherent repetition rate multiplication of a mode-locked laser from 40 MHz to 1 GHz by injection locking.

We have used injection locking to multiply the repetition rate of a passively mode-locked femtosecond fiber laser from 40 MHz to 1 GHz while preserving optical phase coherence between the master laser and the slave output. The system is implemented almost completely in fiber and incorporates gain and passive saturable absorption. The slave repetition rate is set to a rational harmonic of the master repetition rate, inducing pulse formation at the least common multiple of the master and slave repetition rates.

Efficacious, nontoxigenic Bacillus anthracis spore vaccines based on strains expressing mutant variants of lethal toxin components.

We reported previously on the development of a Bacillus anthracis vaccine strain expressing high levels of recombinant protective antigen (rPA) [Cohen et al., Infec Immun 2000;68(8):4549-58]. To further explore the potential of the B. anthracis platform, we generated several attenuated strains expressing lethal toxin components PA and LF, which are biologically inactive, yet retain their antigenic properties. A single injection of 5 x 10(7) spores of one of these strains, carrying PA mutation at a site involved in effector translocation (residues 313-314) was shown to resemble wild type PA in inducing production of high levels of anti-PA neutralizing antibodies and producing effective protective immunity for 12 months. Long-term protection and persistence of functional antibody titers was observed after the gradual elimination of spores from guinea pig tissues 3 months after injection and in the measurable absence of bacteria in tissues. The mutant toxin components could, thus be an effective alternatives to their native counterparts when presented to the immune system in context of a live B. anthracis strain. These live vaccine prototypes may serve as a platform for future multi-component vaccines.

Oral spore vaccine based on live attenuated nontoxinogenic Bacillus anthracis expressing recombinant mutant protective antigen.

An attenuated nontoxinogenic nonencapsulated Bacillus anthracis spore vaccine expressing high levels of recombinant mutant protective antigen (PA), which upon subcutaneous immunization provided protection against a lethal B. anthracis challenge, was found to have the potential to serve also as an oral vaccine. Guinea pigs immunized per os with the recombinant spore vaccine were primed to B. anthracis vegetative antigens as well as to PA, yet only a fraction of the animals (30% to 50%) mounted a humoral response to all of these antigens. Protective immunity provided by per os immunization correlated with a threshold level of PA neutralizing antibody titers and was long-lasting. Protection conferred by per os immunization was attained when the vaccine was administered in the sporogenic form, which, unlike the vegetative cells, survived passage through the gastrointestinal tract. A comparison of immunization of unirradiated spores with immunization of gamma-irradiated spores demonstrated that germination and de novo synthesis of PA were prerequisites for mounting an immune protective response. Oral immunization of guinea pigs with attenuated B. anthracis spores resulted in a characteristic anti-PA immunoglobulin isotype profile (immunoglobulin [G1 IgG1] versus IgG2), as well as induction of specific anti-PA secretory IgA, indicating development of mucosal immunity.

Optimal swimming at low Reynolds numbers.

Efficient swimming at low Reynolds numbers is a major concern of microbots. To compare the efficiencies of different swimmers we introduce the notion of "a swimming drag coefficient" which allows for the ranking of swimmers. We find the optimal swimmer within a certain class of two-dimensional swimmers using conformal mapping techniques.

Optimal rotations of deformable bodies and orbits in magnetic fields.

Deformations can induce rotation with zero angular momentum where dissipation is a natural "cost function." This gives rise to an optimization problem of finding the most effective rotation with zero angular momentum. For certain plastic and viscous media in two dimensions the optimal path is the orbit of a charged particle on a surface of constant negative curvature with a magnetic field whose total flux is half a quantum unit.

Semiclassical analysis and the magnetization of the Hofstadter model.

The magnetization and the de Haas-van Alphen oscillations of Bloch electrons are calculated near commensurate magnetic fluxes. Two phases that appear in the quantization of mixed systems--the Berry phase and a phase first discovered by Wilkinson--play a key role in the theory.

Use of a promoter trap system in Bacillus anthracis and Bacillus subtilis for the development of recombinant protective antigen-based vaccines.

We have recently reported Bacillus anthracis attenuated live vaccine strains efficiently expressing recombinant protective antigen (rPA) and have shown a direct correlation between the level of rPA secreted by these cells and efficacy (S. Cohen, I. Mendelson, Z. Altboum, D. Kobiler, E. Elhanany, T. Bino, M. Leitner, I. Inbar, H. Rosenberg, Y. Gozes, R. Barak, M. Fisher, C. Kronman, B. Velan, and A. Shafferman, Infect. Immun. 68:4549-4558, 2000). To isolate more potent Bacillus promoters for a further increase in the production of rPA, we developed a promoter trap system based on various gfp reporter genes adapted for use in both Bacillus subtilis and B. anthracis backgrounds. Accordingly, a B. anthracis library of 6,000 clones harboring plasmids with chromosomal B. anthracis DNA fragments inserted upstream from gfpuv was constructed. Based on fluorescence intensity, 57 clones carrying potentially strong promoters were identified, some of which were DNA sequenced. The most potent B. anthracis promoter identified (Pntr; 271 bp) was 500 times more potent than the native pagA promoter and 70 times more potent than the alpha-amylase promoter (Pamy). This very potent promoter was tested along with the other promoters (which are three, six, and eight times more potent than Pamy) for the ability to drive expression of rPA in either B. subtilis or B. anthracis. The number of cell-associated pre-PA molecules in B. anthracis was found to correlate well with the strength of the promoter. However, there appeared to be an upper limit to the amount of mature PA secreted into the medium, which did not exceed that driven by Pamy. Furthermore, the rPA constructs fused to the very potent promoters proved to be deleterious to the bacterial hosts and consequently led to genetic instability of the PA expression plasmid. Immunization with attenuated B. anthracis expressing rPA under the control of promoters more potent than Pamy was less efficient in eliciting anti-PA antibodies than that attained with Pamy. The results are consistent with the notion that overexpression of PA leads to severe secretion stress and have practical implications for the design of second-generation rPA-based vaccines.

Search for potential vaccine candidate open reading frames in the Bacillus anthracis virulence plasmid pXO1: in silico and in vitro screening.

A genomic analysis of the Bacillus anthracis virulence plasmid pXO1, aimed at identifying potential vaccine candidates and virulence-related genes, was carried out. The 143 previously defined open reading frames (ORFs) (R. T. Okinaka, K. Cloud, O. Hampton, A. R. Hoffmaster, K. K. Hill, P. Keim, T. M. Koehler, G. Lamke, S. Kumano, J. Mahillon, D. Manter, Y. Martinez, D. Ricke, R. Svensson, and P. J. Jackson, J. Bacteriol. 181:6509-6515, 1999) were subjected to extensive sequence similarity searches (with the nonredundant and unfinished microbial genome databases), as well as motif, cellular location, and domain analyses. A comparative genomics analysis was conducted with the related genomes of Bacillus subtilis, Bacillus halodurans, and Bacillus cereus and the pBtoxis plasmid of Bacillus thuringiensis var. israeliensis. As a result, the percentage of ORFs with clues about their functions increased from approximately 30% (as previously reported) to more than 60%. The bioinformatics analysis permitted identification of novel genes with putative relevance for pathogenesis and virulence. Based on our analyses, 11 putative proteins were chosen as targets for functional genomics studies. A rapid and efficient functional screening method was developed, in which PCR-amplified full-length linear DNA products of the selected ORFs were transcribed and directly translated in vitro and their immunogenicities were assessed on the basis of their reactivities with hyperimmune anti-B. anthracis antisera. Of the 11 ORFs selected for analysis, 9 were successfully expressed as full-length polypeptides, and 3 of these were found to be antigenic and to have immunogenic potential. The latter ORFs are currently being evaluated to determine their vaccine potential.

The renal effect of low-dose dopamine in high-risk patients undergoing coronary angiography.

OBJECTIVES: The purpose of the study was to examine the potential renal protective effect of low-dose dopamine in high-risk patients undergoing coronary angiography. BACKGROUND: Contrast nephropathy is prevalent in patients with chronic renal failure (CRF) and/or diabetes mellitus (DM). Decreased renal blood flow due to vasoconstriction was suggested as a contributory mechanism. Low-dose dopamine has a dilatory effect on the renal vasculature. METHODS: Sixty-six patients with mild or moderate CRF and/or DM undergoing coronary angiography were prospectively double-blindedly randomized, to either 120 ml/day of 0.9% saline plus dopamine 2 microg/kg/min (Dopamine group) or saline alone (Control group) for 48 h. RESULTS: Thirty-three Dopamine-treated (30 diabetics and 6 with CRF) and 33 Control (28 diabetics and 5 with CRF) patients were compared. Plasma creatinine (Cr) level increased in the Control group from 100.6+/-5.2 before to 112.3+/-8.0 micromol/liter within five days after angiography (p = 0.003), and in the Dopamine group from 100.3+/-5.4 before to 117.5+/-8.8 micromol/liter after angiography (p = 0.0001), respectively. There was no significant difference in the change of Cr level (deltaCr) between the two groups. However, in a subgroup of patients with peripheral vascular disease (PVD), deltaCr was -2.4+/-2.3 in the Control group and 30.0+/-12.0 micromol/liter in the Dopamine group (p = 0.01). No significant difference occurred in deltaCr between Control and Dopamine in subgroups of patients with preangiographic CRF or DM. CONCLUSIONS: Contrast material caused a small but significant increase in Cr blood level in high-risk patients. There is no advantage of dopamine over adequate hydration in patients with mild to moderate renal failure or DM undergoing coronary angiography. Dopamine should be avoided in patients with PVD exposed to contrast medium.

The glucuronic acid utilization gene cluster from Bacillus stearothermophilus T-6.

A lambda-EMBL3 genomic library of Bacillus stearothermophilus T-6 was screened for hemicellulolytic activities, and five independent clones exhibiting beta-xylosidase activity were isolated. The clones overlap each other and together represent a 23.5-kb chromosomal segment. The segment contains a cluster of xylan utilization genes, which are organized in at least three transcriptional units. These include the gene for the extracellular xylanase, xylanase T-6; part of an operon coding for an intracellular xylanase and a beta-xylosidase; and a putative 15.5-kb-long transcriptional unit, consisting of 12 genes involved in the utilization of alpha-D-glucuronic acid (GlcUA). The first four genes in the potential GlcUA operon (orf1, -2, -3, and -4) code for a putative sugar transport system with characteristic components of the binding-protein-dependent transport systems. The most likely natural substrate for this transport system is aldotetraouronic acid [2-O-alpha-(4-O-methyl-alpha-D-glucuronosyl)-xylotriose] (MeGlcUAXyl3). The following two genes code for an intracellular alpha-glucuronidase (aguA) and a beta-xylosidase (xynB). Five more genes (kdgK, kdgA, uxaC, uxuA, and uxuB) encode proteins that are homologous to enzymes involved in galacturonate and glucuronate catabolism. The gene cluster also includes a potential regulatory gene, uxuR, the product of which resembles repressors of the GntR family. The apparent transcriptional start point of the cluster was determined by primer extension analysis and is located 349 bp from the initial ATG codon. The potential operator site is a perfect 12-bp inverted repeat located downstream from the promoter between nucleotides +170 and +181. Gel retardation assays indicated that UxuR binds specifically to this sequence and that this binding is efficiently prevented in vitro by MeGlcUAXyl3, the most likely molecular inducer.

Cloning and DNA sequence of the gene coding for Bacillus stearothermophilus T-6 xylanase.

Bacillus stearothermophilus T-6 produces an extracellular thermostable xylanase. Affinity-purified polyclonal serum raised against the enzyme was used to screen a genomic library of B. stearothermophilus T-6 constructed in lambda-EMBL3. Two positive phages were isolated, both containing similar 13-kb inserts, and their lysates exhibited xylanase activity. A 3,696-bp SalI-BamHI fragment containing the xylanase gene was subcloned in Escherichia coli and subsequently sequenced. The open reading frame of xylanase T-6 consists of 1,236 bp. On the basis of sequence similarity, two possible -10 and -35 regions, a ribosome-binding site at the 5' end of the gene and a potential transcriptional termination motif at the 3' end of the gene, were identified. From the previously known N-terminal amino acid sequence of xylanase T-6 and the possible ribosome-binding site, a putative 28-amino-acid signal peptide was deduced. The mature xylanase T-6 consists of 379 amino acids with a calculated molecular weight and pI of 43,808 and 6.88, respectively. Multiple alignment of beta-glycanase amino acid sequences revealed highly conserved regions. Northern (RNA) blot analysis indicated that the xylanase T-6 transcript is about 1.4 kb and that the induction of this enzyme synthesis by xylose is on the transcriptional level.

Adherence of Mycoplasma gallisepticum to glass.

Attachment of washed Mycoplasma gallisepticum cells to glass was quantified with organisms in which membrane lipids were labelled with 3H. Siliconization of the test tubes decreased attachment, while centrifugation increased it. Attachment increased with temperature, decreased with increasing pH and ionic strength of the attachment mixture, but was unaffected by Ca2+, Mg2+ and EDTA. This suggests that ionic bonds, but not salt bridges, participate in the attachment process. Glycophorin, the major receptor responsible for M. gallisepticum attachment to erythrocytes, partially inhibited the attachment of the organisms to glass. However, bovine serum albumin also decreased attachment. Extensive pretreatment of the organisms with trypsin decreased their ability to attach to glass by about 35 to 40%. Trypsin and pronase failed to detach the organisms already bound to glass, suggesting that external mycoplasma cell components, other than membrane proteins, also participate in attachment of the organisms to glass.