Sizing sub-diffraction limit electrosprayed droplets by structured illumination microscopy.

Electrosprayed droplets are widely studied for their role in the formation of ions at atmospheric pressure. Most droplet measurement methods used today employ light scattering to infer information about an electrosprayed droplet's size. However, these methods fail to measure droplets smaller than about 400 nm in diameter due to constraints imposed by the diffraction limit of light. To overcome this limitation, a super resolution fluorescence microscopy-based method for determining the sizes of electrosprayed droplets has been developed. Solutions containing rhodamine B and different amounts of glycerol were paper sprayed and nanoelectrosprayed onto conductive microscope coverslips using a single, high voltage pulse. Images of the deposited droplets were collected using a super resolution microscope operating in 3D structured illumination microscopy mode (3D-SIM). The sizes of droplets were measured using a modified circular Hough transformation program in Matlab. On average, the diameters of paper sprayed droplets were between 500 nm and 2 µm while almost all nanoelectrosprayed droplets were smaller than 1 µm. The center of a paper spray plume exhibited larger droplets than those at the periphery, likely due to greater Coulombic repulsive forces acting on the smaller droplets to drive them outwards. The periphery also likely contained progeny droplets in addition to smaller parent droplets. It was possible to alter the sizes of nanoelectrosprayed droplets in several ways, including by changing the solvent composition and voltage applied to the emitter. Droplets consisting of high concentrations of glycerol were larger than droplets containing high concentrations of methanol, presumably due to the high surface tension of glycerol. Correspondingly, droplets became smaller when the voltage applied to the emitter was increased, likely due to the ability to overcome the surface tension of the solvent more easily. The smallest detectable droplets confidently measured with this method were 200 nm in diameter. This method demonstrates a new way of measuring the sizes of electrosprayed droplets with half the diameter of conventional droplet size measurement methods. Through further optimization, it may be possible to measure the sizes of electrosprayed droplets as small as the theoretical resolution limit of SIM (∼100 nm).

Normal and abnormal heme biosynthesis. Part 7. Synthesis and metabolism of coproporphyrinogen-III analogues with acetate or butyrate side chains on rings C and D. Development of a modified model for the active site of coproporphyrinogen oxidase.

Analogues of coproporphyrinogen-III have been prepared with acetate or butyrate groups attached to the C and D pyrrolic subunits. The corresponding porphyrin methyl esters were synthesized by first generating a,c-biladienes by reacting a dipyrrylmethane with pyrrole aldehydes in the presence of HBr. Cyclization with copper(II) chloride in DMF, followed by demetalation with 15% H(2)SO(4)-TFA and reesterification, gave the required porphyrins in excellent yields. Hydrolysis with 25% hydrochloric acid and reduction with sodium-amalgam gave novel diacetate and dibutyrate porphyrinogens 9. Diacetate 9a was incubated with chicken red cell hemolysates (CRH), but gave complex results due to the combined action of two of the enzymes present in these preparations. Separation of uroporphyrinogen decarboxylase (URO-D) from coproporphyrinogen oxidase (CPO) allowed the effects of both enzymes on the diacetate substrate to be assessed. Porphyrinogen 9a proved to be a relatively poor substrate for CPO compared to the natural substrate coproporphyrinogen-III, and only the A ring propionate moiety was processed to a significant extent. Similar results were obtained for incubations of 9a with purified human recombinant CPO. Diacetate 9a was also a substrate for URO-D and a porphyrinogen monoacetate was the major product in this case; however, some conversion of a second acetate unit was also evident. The dibutyrate porphyrinogen 9b was only recognized by the enzyme CPO, but proved to be a modest substrate for incubations with CRH. However, 9b was an excellent substrate for purified human recombinant CPO. The major product for these incubations was a monovinylporphyrinogen, but some divinyl product was also generated in incubations using purified recombinant human CPO. The incubation products were converted into the corresponding porphyrin methyl esters, and these were characterized by proton NMR spectroscopy and mass spectrometry. The results extend our understanding of substrate recognition and catalysis for this intriguing enzyme and have allowed us to extend the active site model for CPO. In addition, the competitive action of both URO-D and CPO on the same diacetate porphyrinogen substrate provides additional perspectives on the potential existence of abnormal pathways for heme biosynthesis.

Nested uncertainties in biochemical models.

Dynamic modelling of biochemical reaction networks has to cope with the inherent uncertainty about biological processes, concerning not only data and parameters but also kinetics and structure. These different types of uncertainty are nested within each other: uncertain network structures contain uncertain reaction kinetics, which in turn are governed by uncertain parameters. Here, the authors review some issues arising from such uncertainties and sketch methods, solutions and future directions to deal with them.

Pseudomonas aeruginosa forms biofilms in acute infection independent of cell-to-cell signaling.

Biofilms are bacterial communities residing within a polysaccharide matrix that are associated with persistence and antibiotic resistance in chronic infections. We show that the opportunistic pathogen Pseudomonas aeruginosa forms biofilms within 8 h of infection in thermally injured mice, demonstrating that biofilms contribute to bacterial colonization in acute infections as well. Using light, electron, and confocal scanning laser microscopy, P. aeruginosa biofilms were visualized within burned tissue surrounding blood vessels and adipose cells. Although quorum sensing (QS), a bacterial signaling mechanism, coordinates differentiation of biofilms in vitro, wild-type and QS-deficient P. aeruginosa strains formed similar biofilms in vivo. Our findings demonstrate that P. aeruginosa forms biofilms on specific host tissues independently of QS.

Diversity of biofilms produced by quorum-sensing-deficient clinical isolates of Pseudomonas aeruginosa.

The quorum-sensing (QS) systems control several virulence attributes of Pseudomonas aeruginosa. Five QS-deficient P. aeruginosa clinical isolates (CI) that were obtained from wound (CI-1), tracheal (CI-2, CI-3, CI-4) and urinary tract (CI-5) infections had previously been characterized. In this study, a flow-through continuous-culture system was utilized to examine in detail the biofilms formed by these isolates in comparison with the P. aeruginosa prototrophic strain PAO1. Analysis of the biofilms by confocal laser scanning microscopy and COMSTAT image analysis at 1 and 7 days post-inoculation showed that the isolates produced diverse biofilms. In comparison with PAO1, the CI produced biofilms that scarcely or partially covered the surface at day 1, although CI-1 produced larger microcolonies. At day 7, CI-2 and CI-4 produced mature biofilms denser than that produced by PAO1, while the biofilm formed by CI-1 changed very little from day 1. CI-1 was defective in both swarming and twitching motilities, and immunoblotting analysis confirmed that it produced a reduced level of PilA protein. The twitching-motility defect of CI-1 was not complemented by a plasmid carrying intact pilA. In the 48 h colony biofilm assay, the CI varied in susceptibility to imipenem, gentamicin and piperacillin/tazobactam. These results suggest that: (1) the isolates produced biofilms with different structures and densities from that of PAO1; (2) biofilm formation by the isolates was not influenced by either the isolation site or the QS deficiencies of the isolates; (3) the behaviour of CI-1 in the different biofilm systems may be due to its lack of swarming motility and type IV pilus-related twitching motility.

Tempo and mode of early gene loss in endosymbiotic bacteria from insects.

BACKGROUND: Understanding evolutionary processes that drive genome reduction requires determining the tempo (rate) and the mode (size and types of deletions) of gene losses. In this study, we analysed five endosymbiotic genome sequences of the gamma-proteobacteria (three different Buchnera aphidicola strains, Wigglesworthia glossinidia, Blochmannia floridanus) to test if gene loss could be driven by the selective importance of genes. We used a parsimony method to reconstruct a minimal ancestral genome of insect endosymbionts and quantified gene loss along the branches of the phylogenetic tree. To evaluate the selective or functional importance of genes, we used a parameter that measures the level of adaptive codon bias in E. coli (i.e. codon adaptive index, or CAI), and also estimates of evolutionary rates (Ka) between pairs of orthologs either in free-living bacteria or in pairs of symbionts. RESULTS: Our results demonstrate that genes lost in the early stages of symbiosis were on average less selectively constrained than genes conserved in any of the extant symbiotic strains studied. These results also extend to more recent events of gene losses (i.e. among Buchnera strains) that still tend to concentrate on genes with low adaptive bias in E. coli and high evolutionary rates both in free-living and in symbiotic lineages. In addition, we analyzed the physical organization of gene losses for early steps of symbiosis acquisition under the hypothesis of a common origin of different symbioses. In contrast with previous findings we show that gene losses mostly occurred through loss of rather small blocks and mostly in syntenic regions between at least one of the symbionts and present-day E. coli. CONCLUSION: At both ancient and recent stages of symbiosis evolution, gene loss was at least partially influenced by selection, highly conserved genes being retained more readily than lowly conserved genes: although losses might result from drift due to the bottlenecking of endosymbiontic populations, we demonstrated that purifying selection also acted by retaining genes of greater selective importance.

Analysis of quorum sensing-deficient clinical isolates of Pseudomonas aeruginosa.

Pseudomonas aeruginosa produces multiple virulence factors and causes different types of infections. Previous clinical studies identified P. aeruginosa isolates that lack individual virulence factors. However, the impact of losing several virulence factors simultaneously on the in vivo virulence of P. aeruginosa is not completely understood. The P. aeruginosa cell-to-cell communication system, or quorum sensing (QS), controls the production of several virulence factors. Animal studies using constructed QS mutants indicated that loss of the QS system severely impacts the virulence of P. aeruginosa. In this study, we tried to determine if deficiency within the QS system compromises the ability of P. aeruginosa to establish infections in humans. We have identified five QS-deficient strains through screening 200 isolates from patients with urinary tract, lower respiratory tract and wound infections. These strains lacked LasB and LasA activities and produced either no or very low levels of the autoinducers N-(3-oxododecanoyl) homoserine lactone and N-butyryl homoserine lactone. PCR analysis revealed that three isolates contained all four QS genes (lasI, lasR, rhlI and rhlR) while two isolates lacked both the lasR and rhlR genes. We also examined the five isolates for other virulence factors. The isolates produced variable levels of exotoxin A and, with one exception, were deficient in pyocyanin production. One isolate produced the type III secretion system (TTSS) effector proteins ExoS and ExoT, two isolates produced ExoT only and two isolates produced no TTSS proteins. The isolates produced weak to moderate biofilms on abiotic surfaces. Analysis of the patients' data revealed that two of the isolates represented a single strain that was isolated twice from the same patient within a 1 month interval. One QS-deficient clinical isolate (CI-1) lacked all tested virulence factors and produced a weak biofilm. These results suggest that naturally occurring QS-deficient strains of P. aeruginosa do occur and are capable of causing infections; and, that besides the known virulence factors, additional factors may contribute to the ability of certain strains such as CI-1 to establish an infection.

Pulmonary Ureaplasma urealyticum is associated with the development of acute lung inflammation and chronic lung disease in preterm infants.

Previously, we have reported marked pulmonary inflammation in infants who develop chronic lung disease of prematurity. We revisited these infants who did not have clinical or laboratory evidence of infection and searched for Ureaplasma urealyticum, group B streptococci, and other microbes by reverse transcription-PCR performed on RNA extracted from 93 bronchoalveolar lavage samples. From infants ventilated for respiratory distress syndrome, 6 (gestation, 28 wk; birthweight, 880 g) were positive for U. urealyticum and 11 (25 wk, 800 g) were negative. Five (83%) positive and four (36%) negative infants developed chronic lung disease. Each infant was colonized with either biovar 1 or biovar 2 but not both. U. urealyticum was very weakly detectable in two infants on d 1 but was detected in five of six infants at d 10. Furthermore, pulmonary neutrophils, alveolar macrophages, soluble intercellular adhesion molecule-1, and IL-1beta on d 10 and IL-6 and IL-8 at d 1 were significantly increased in the positive group. A variety of organisms were identified in six samples between 14 and 21 d of age, but all samples were negative for group B streptococci. Our data suggest that U. urealyticum colonization is associated with the development of pulmonary inflammation in infants who subsequently develop chronic lung disease.

Prolactin activates Stat1 but does not antagonize Stat1 activation and growth inhibition by type I interferons in human breast cancer cells.

Type I interferons (IFN alpha and IFN beta) are presently used in the adjuvant treatment of several human cancers. However, these cytokines have demonstrated only modest success in breast cancer therapy, and research efforts have focused on improving their efficacy. Recent progress in understanding the molecular mechanisms underlying the antiproliferative effects of IFNs has identified the cytoplasmic transcription factor Stat1 as a critical mediator. It is, therefore, possible that IFN-induced growth inhibition of mammary epithelial cells is counteracted by other cytokines that also use Stat1. One such candidate IFN-antagonist with particular relevance to breast cancer is the mammotropic hormone prolactin (PRL). The main goal of this study was to examine whether PRL would interfere with type I IFN (IFN alpha/beta) signal transduction by competing for limited cytoplasmic Stat factors. A second aim was to test whether pretreatment of mammary tumor cell lines with IFN gamma could enhance the effect of IFN alpha/beta. By analyzing the effect of PRL on IFN alpha/beta-induced tyrosine phosphorylation of Stat proteins and their binding to IFN-regulated genes, we now report that costimulation of PRL receptors did not interfere with IFN alpha/beta signals in several human breast cancer cell lines, including T47D, MCF-7, and BT-20. Specifically, PRL did not affect IFN alpha/beta-induced tyrosine phosphorylation or heterodimerization of Stat1 and Stat2 in any cell line. Instead, IFN alpha/beta- and PRL-induced tyrosine phosphorylation of Stat1 was additive and occurred without evidence of competition for limited concentrations of cytoplasmic Stat1. A similar additive relationship was observed on IFN alpha/beta- and PRL-induced Stat3 tyrosine phosphorylation. Furthermore, electrophoretic mobility shift assays showed that type I IFNs induced predominantly Stat1-Stat2 or Stat1-Stat3 heteromeric complexes with various IFN-response elements of IFN-stimulated genes, whereas PRL induced Stat1 homodimers. Despite significant mutual use of Stats by IFNs and PRL, these results indicated a high degree of signaling specificity in the two receptor systems, and that cytoplasmic levels of Stat proteins were not limiting. Similarly, PRL did not interfere with the growth-inhibitory effect of IFN beta. On the other hand, the study indicated that pretreatment of human breast cancer cell lines with IFN gamma enhanced the growth-inhibitory action of type I IFNs, suggesting a possible avenue for improving the effect of type I IFNs in the treatment of breast cancer patients.

Molecular size of the human platelet alpha 2-adrenergic receptor as determined by radiation inactivation.

The size of the alpha 2-adrenergic receptor in human platelet membranes has been determined by radiation inactivation/target size analysis to be 160,000 daltons. The size of the platelet alpha 2-receptors is essentially identical to that of the alpha 1-adrenergic receptor of the rat liver membrane.

Molecular properties of the slow inward calcium channel. Molecular weight determinations by radiation inactivation and covalent affinity labeling.

The slow inward calcium channel, identified by physiologic and pharmacologic responses and [3H]nitrendipine-specific binding, has been characterized by radiation inactivation and covalent affinity labeling. Target size analysis of guinea pig ileum longitudinal smooth muscle membranes indicates a molecular weight of 278,000 for the calcium channel. An affinity label analog of nifedipine and nitrendipine, 2,6-dimethyl-3,5-dicarbomethoxy-4-(2-isothiocyanatophenyl)-1,4-dihydropyridine, was found to inhibit the calcium channel by a covalent interaction with a protein subunit (Mr = 45,000) of the calcium channel.

Purification and molecular characterization of neurotransmitter receptors.

These data indicate a number of similarities between the neurotransmitter receptors of different pharmacological classes. We are pursuing the hypothesis that the neurotransmitter receptors may have evolved from one another and contain constant regions (eg, adenylate cyclase and calcium channel interaction sites) and variable regions (eg, neurotransmitter- or hormone-binding sites). It is clear that monoclonal antibodies are keys to this end.