Preoperative topical liposomal ozone dispersion to reduce bacterial colonization in conjunctival sac and periocular skin: Preliminary study in dogs.

Prophylaxis represents a keystone to reduce periocular skin and ocular conjunctiva bacterial load before surgical procedures. Despite many prophylactic agents are available the preferred perioperative ocular surface antimicrobial is still unknown. The purpose of this study was to assess the effectiveness of preoperative liposomal ozone dispersion in reducing bacterial colonization from the conjunctival sac and periocular skin in dogs, in comparison with povidone-iodine and fluoroquinolone. Twenty-two owned dogs consisting with 44 eyes in total scheduled for ophthalmic surgical procedure were enrolled for the study and divided in four groups receiving either ozone dispersion or povidone iodine in eyelid and conjunctiva, fluoroquinolone or placebo. A swab was taken before and after the antisepsis protocol evaluating total microbial count, coagulase positive and negative staphylococci. Statistical analysis revealed a significant decrease in colony forming units (CFU) for total microbial count, coagulase positive and negative staphylococci both for liposomal ozone dispersion and povidone iodine. No statistical differences were detected in median CFU for both one-day placebo and fluoroquinolone preoperative prophylactic topical therapy. The results of this preliminary study demonstrate that liposomal ozone-dispersion is as effective as povidone iodine to reduce preoperative bacterial load in ocular surface.

Thrombin cleaves and activates the protease-activated receptor 2 dependent on thrombomodulin co-receptor availability.

INTRODUCTION: Protease-activated receptors (PARs) evolved to react to extracellular proteolytic activity. In mammals, three of the four PARs (PAR1, PAR3, and PAR4) that are expressed respond to the prototypical procoagulant enzyme thrombin, whereas PAR2 was assumed to resist activation by thrombin. To date, involvement of cell surface thrombin-recruiting co-receptors such as thrombomodulin (TM), which potentially facilitates PAR2 cleavage, has not been addressed. Thus, we examined whether TM-bound thrombin cleaved PAR2 and tested biological responses such as nuclear factor kappa B (NF-κB) DNA binding activity and cytokine release. MATERIALS AND METHODS: We examined 293T cells overexpressing PAR2 and TM for thrombin recruitment by TM promoting PAR2 cleavage. To test for the TM-thrombin interactions required for PAR2 cleavage and to map cleavage sites on PAR2, mutant constructs of TM or PAR2 were engineered. Biological effects because of PAR2 activation were investigated using an NF-κB reporter system and cytokine release. RESULTS AND CONCLUSIONS: We identified that, at low to moderate concentrations, thrombin cleaved PAR2 in a TM co-receptor-dependent manner with cleavage efficiency comparable to that of trypsin. In TM's presence, thrombin efficiently cleaved both, PAR1 and PAR2, albeit kinetics differed. Whereas the majority of surface expressed PAR1 was immediately cleaved off, prolonged exposure to thrombin resulted in few additional cleavage. In contrast, PAR2 cleavage was sustained upon prolonged exposure to thrombin. However, TM EGF-like domain 5 was required and TM chondroitin sulfate (CS) proteoglycan sites serine 490 and serine 492 assisted in PAR2 cleavage, while thrombin preferentially cleaved at arginine 36 on PAR2's N-terminus. Note that thrombin-induced activation of NF-κB via PAR2 resulted in release of interleukin-8. Thus, we provide a novel concept of how thrombin efficiently cleaves PAR2 in a TM-dependent manner, resulting in pro-inflammatory interleukin-8 release. This unexpected pro-inflammatory role of TM, promoting cleavage and activation of PAR2 by thrombin, may lead to novel therapeutic options for treating inflammatory and malignant diseases.

Effect of Global Regulators RpoS and Cyclic-AMP/CRP on the Catabolome and Transcriptome of Escherichia coli K12 during Carbon- and Energy-Limited Growth.

For heterotrophic microbes, limited availability of carbon and energy sources is one of the major nutritional factors restricting the rate of growth in most ecosystems. Physiological adaptation to this hunger state requires metabolic versatility which usually involves expression of a wide range of different catabolic pathways and of high-affinity carbon transporters; together, this allows for simultaneous utilization of mixtures of carbonaceous compounds at low concentrations. In Escherichia coli the stationary phase sigma factor RpoS and the signal molecule cAMP are the major players in the regulation of transcription under such conditions; however, their interaction is still not fully understood. Therefore, during growth of E. coli in carbon-limited chemostat culture at different dilution rates, the transcriptomes, expression of periplasmic proteins and catabolomes of strains lacking one of these global regulators, either rpoS or adenylate cyclase (cya), were compared to those of the wild-type strain. The inability to synthesize cAMP exerted a strong negative influence on the expression of alternative carbon source uptake and degradation systems. In contrast, absence of RpoS increased the transcription of genes belonging to high-affinity uptake systems and central metabolism, presumably due to reduced competition of σ(D) with σ(S). Phenotypical analysis confirmed this observation: The ability to respire alternative carbon substrates and to express periplasmic high-affinity binding proteins was eliminated in cya and crp mutants, while these properties were not affected in the rpoS mutant. As expected, transcription of numerous stress defence genes was negatively affected by the rpoS knock-out mutation. Interestingly, several genes of the RpoS stress response regulon were also down-regulated in the cAMP-negative strain indicating a coordinated global regulation. The results demonstrate that cAMP is crucial for catabolic flexibility during slow, carbon-limited growth, whereas RpoS is primarily involved in the regulation of stress response systems necessary for the survival of this bacterium under hunger conditions.

Methane dynamics in an alpine fen: a field-based study on methanogenic and methanotrophic microbial communities.

Wetlands are important sources of the greenhouse gas methane (CH4). We provide an in situ study of CH4 dynamics in the permanently submerged soil of a Swiss alpine fen. Physico-chemical pore water analyses were combined with structural and microbiological analyses of soil cores at high vertical resolution down to 50 cm depth. Methanotrophs and methanogens were active throughout the depth profile, and highest abundance of active methanotrophs and methanogens [6.1 × 10(5) and 1.1 × 10(7) pmoA and mcrA transcripts (g soil)(-1), respectively] was detected in the uppermost 2 cm of the soil. Active methanotrophic communities in the near-surface zone, dominated by viable mosses, varied from the communities in the deeper zones, but further changes with depth were not pronounced. Apart from a distinct active methanogenic community in the uppermost sample, a decrease of acetoclastic Methanosaetaceae with depth was observed in concomitance with decreasing root surface area. Overall, root surface area correlated with mcrA transcript abundance and CH4 pore water concentrations, which peaked (137.1 µM) at 10 to 15 cm depth. Our results suggest that stimulation of methanogenesis by root exudates of vascular plants had a stronger influence on CH4 dynamics than stimulation of CH4 oxidation by O2 input.

Analysis of structure, function, and activity of a benzene-degrading microbial community.

We identified phylotypes performing distinct functions related to benzene degradation in complex microbial biofilms from an aerated treatment pond containing coconut textile. RNA- and protein-stable isotope probing (SIP) and compound-specific stable isotope analysis were applied to delineate bacteria and predominant pathways involved in the degradation of benzene. In laboratory microcosms, benzene was degraded at rates of ≥ 11 µM per day and per gram coconut textile under oxic conditions. Carbon isotope fractionation with isotopic enrichment factors (ε) of -0.6 to -1‰ and no significant hydrogen isotope fractionation indicated a dihydroxylation reaction for the initial ring attack. The incubation with [(13)C6]-benzene led to (13)CO2 formation accompanied by (13)C-labeling of RNA and proteins of the active biomass. Phylogenetic analysis of the (13)C-labeled RNA revealed that phylotypes related to Zoogloea, Ferribacterium, Aquabacterium, and Hydrogenophaga within the Betaproteobacteria predominantly assimilated carbon from benzene. Although the phylogenetic classification of identified (13)C-labeled proteins was biased by the incomplete metagenome information of public databases, it matched with RNA-SIP results at genus level. The detection of (13)C-labeled proteins related to toluene dioxygenase and catechol 2,3-dioxygenase suggests benzene degradation by a dihydroxylation pathway with subsequent meta-cleavage of formed catechol.

Freeze-coring method for characterization of microbial community structure and function in wetland soils at high spatial resolution.

A simple freeze-coring method was developed to obtain structurally intact cores from wetland soils. A copper tube was inserted into the wetland and filled with ethanol and dry ice to freeze the surrounding soil. Biological structure and function could be analyzed, and labile compounds such as mRNA were recovered.

Assimilation of benzene carbon through multiple trophic levels traced by different stable isotope probing methodologies.

The flow of benzene carbon along a food chain consisting of bacteria and eukaryotes, including larvae (Diptera: Chironomidae), was evaluated by total lipid fatty acids (TLFAs)-, amino acid- and protein-stable isotope probing (SIP). A coconut-fibre textile, colonized by a benzene-degrading biofilm, was sampled in a system established for the remediation of benzene, toluene, ethylbenzene and xylenes (BTEX)-polluted groundwater and incubated with (12)C- and [(13)C(6)]-benzene (>99 at.%) in a batch-scale experiment for 2-8 days. After 8 days, Chironomus sp. larvae were added to study carbon flow to higher trophic levels. Gas chromatography-combustion-isotope ratio monitoring mass spectrometry of TLFA showed increased isotope ratios in the (13)C-benzene-incubated biofilm. A higher (13)C-enrichment was observed in TLFAs, indicative of Gram-negative bacteria than for Gram-positive. Fatty acid indicators of eukaryotes showed significant (13)C-incorporation, but to a lower extent than bacterial indicators. Fatty acids extracted from larvae feeding on (13)C-biofilm reached an isotopic ratio of 1.55 at.%, illustrating that the larvae feed, to some extent, on labelled biomass. No (13)C-incorporation was detectable in larval proteins after their separation by sodium-dodecyl sulphate-polyacrylamide gel electrophoresis and analysis by nano-liquid-chromatography-mass spectrometry. The flow of benzene-derived carbon could be traced in a food web consisting of bacteria and eukaryotes.

Elucidating MTBE degradation in a mixed consortium using a multidisciplinary approach.

The structure and function of a microbial community capable of biodegrading methyl-tert-butyl ether (MTBE) was characterized using compound-specific stable isotope analysis (CSIA), clone libraries and stable isotope probing of proteins (Protein-SIP). The enrichment culture (US3-M), which originated from a gasoline-impacted site in the United States, has been enriched on MTBE as the sole carbon source. The slope of isotopic enrichment factors (epsilon(C) of -2.29+/-0.03 per thousand; epsilon(H) of -58+/-6 per thousand) for carbon and hydrogen discrimination (Deltadelta(2)H/Deltadelta(13)C) was on average equal to Lambda=24+/-2, a value closely related to the reaction mechanism of MTBE degradation in Methylibium petroleiphilum PM1. 16S rRNA gene libraries revealed sequences belonging to M. petroleiphilum PM1, Hydrogenophaga sp., Thiothrix unzii, Rhodobacter sp., Nocardiodes sp. and different Sphingomonadaceae bacteria. Protein-SIP analysis of the culture grown on (13)C-MTBE as the only carbon source revealed that proteins related to members of the Comamonadaceae family, such as Delftia acidovorans, Acidovorax sp. or Comamonas sp., were not (13)C-enriched, whereas proteins related to M. petroleiphilum PM1 showed an average incorporation of 94.5 atom%(13)C. These results indicate a key role for this species in the degradation of MTBE within the US3-M consortia. The combination of CSIA, molecular biology and Protein-SIP facilitated the analysis of an MTBE-degrading mixed culture from a functional and phylogenetic point of view.

Aerated treatment pond technology with biofilm promoting mats for the bioremediation of benzene, MTBE and ammonium contaminated groundwater.

A novel aerated treatment pond for enhanced biodegradation of groundwater contaminants was tested under field conditions. Coconut fibre and polypropylene textiles were used to encourage the development of contaminant-degrading biofilms. Groundwater contaminants targeted for removal were benzene, methyl tert-butyl ether (MTBE) and ammonium. Here, we present data from the first 14 months of operation and compare contaminant removal rates, volatilization losses, and biofilm development in one pond equipped with coconut fibre to another pond with polypropylene textiles. Oxygen concentrations were constantly monitored and adjusted by automated aeration modules. A natural transition from anoxic to oxic zones was simulated to minimize the volatilization rate of volatile organic contaminants. Both ponds showed constant reductions in benzene concentrations from 20 mg/L at the inflow to about 1 microg/L at the outflow of the system. A dynamic air chamber (DAC) measurement revealed that only 1% of benzene loss was due to volatilization, and suggests that benzene loss was predominantly due to aerobic mineralization. MTBE concentration was reduced from around 4 mg/L at the inflow to 3.4-2.4 mg/L in the system effluent during the first 8 months of operation, and was further reduced to 1.2 mg/L during the subsequent 6 months of operation. Ammonium concentrations decreased only slightly from around 59 mg/L at the inflow to 56 mg/L in the outflow, indicating no significant nitrification during the first 14 months of continuous operation. Confocal laser scanning microscopy (CLSM) demonstrated that microorganisms rapidly colonized both the coconut fibre and polypropylene textiles. Microbial community structure analysis performed using denaturing gradient gel electrophoresis (DGGE) revealed little similarity between patterns from water and textile samples. Coconut textiles were shown to be more effective than polypropylene fibre textiles for promoting the recruitment and development of MTBE-degrading biofilms. Biofilms of both textiles contained high numbers of benzene metabolizing bacteria suggesting that these materials provide favourable growth conditions for benzene degrading microorganisms.

Sequence specific primer extension RNA analysis (SeSPERA) for the investigation of substrate utilization of microbial communities.

A simple, fast and cost-effective method for the taxon-specific separation of various 16S rRNAs was developed. This primer extension mediated separation of different RNA species was obtained on agarose gels. The method provides an easy way for the investigation of substrate mediated radioisotope incorporation into the RNA.

Induction of the yjbEFGH operon is regulated by growth rate and oxygen concentration.

The yjbEFGH operon of Escherichia coli was previously shown to be involved in exopolysaccharide production and induced during biofilm formation by osmotic stress; in this communication we investigate some of the factors taking part in its regulation. We show that induction of yjbF'::luxCDABE transcriptional fusion in response to osmotic shock is limited to the early growth phase of batch-grown cells, and is dictated by cell density in an oxygen-related manner: induction was maximal at low cell density and at high oxygen concentrations. The dependency on cell density and oxygen availability was verified in both batch and continuous culture, using either bioluminescence (luxCDABE) or beta-galactosidase (lacZ) genes as the reporter system. It is further shown that yjb is also induced by low specific growth rates, even in the absence of osmotic stress. Finally, it is demonstrated that induction of the yjbEFGH operon, though clearly a stress response, is not a part of the general stress regulon under the positive control of RpoS, as it is negatively affected by this sigma factor.

Compromise between spherical and chromatic aberration and depth of focus in aspheric intraocular lenses.

PURPOSE: To evaluate the performance of 4 aspheric intraocular lenses (IOLs) in scotopic lighting conditions to determine the IOL with the best image quality at different spherical and chromatic aberrations and depths of focus. SETTING: Eye Institute, Optic Physiopathological Department, University of Florence, Florence, Italy. METHODS: Four IOLs were tested. In the Tecnis (Advanced Medical Optics, Inc.), IQ (Alcon Surgical), and KS-3Ai (Staar Surgical) IOLs, the aspheric profile is on the anterior surface of the optic. In the SofPort IOL (Bausch & Lomb), it is on the anterior and posterior surfaces and is preloaded in the injector. Spot diagrams were used to capture light dispersion, monochromatic point-spread function (PSF) to evaluate the light distribution on the fovea of the eye model, and monochromatic modulation transfer function (MTF) to evaluate image contrast. Depth of focus was evaluated at best focus and -0.08 mm and +0.08 mm from best focus. RESULTS: The spot diagrams for the Tecnis IOL were well-focused within a diameter of 10 microm. For the other aspheric IOLs, the pattern was broader, indicating greater dispersion and larger chromatic aberrations. The monochromatic PSF and Strehl ratios favored the Tecnis lens, demonstrating that light energy is focused on the area of the eye model that represents the fovea. The monochromatic MTF showed higher contrast levels for the Tecnis lens than the other IOLs at yellow and blue wavelengths, but the KS-3Ai had higher contrast at the red frequency. The depth of focus testing showed overlapping of the yellow and blue frequencies and partial overlapping of the yellow, blue, and red for the Tecnis lens, indicating good depth of focus. The IQ IOL had higher luminance but less overlap. The SofPort and KS-3Ai IOLs had lower luminance thresholds. CONCLUSIONS: It is possible to find the compromise in which residuals of spherochromatic aberration enhance the depth of focus by decreasing the image sharpness or the amount of light on the fovea. The Tecnis IOL provided the best compromise between spherical and chromatic aberrations and depth of focus.

Comparative assessment of contrast with spherical and aspherical intraocular lenses.

PURPOSE: To analyze the contrast differences between spherical and aspherical intraocular lenses (IOLs) using polychromatic modulation transfer function (MTF) computations. SETTING: Oto-Neuro-Ophthalmological Department, Eye Institute, University of Florence, Florence, Italy. METHODS: A questionnaire evaluating patients' subjective perception of halos, glare, and contrast sensitivity in different lighting conditions was administered to 2 groups of pseudophakic patients. In the first group, a spherical IOL (AR40e, AMO) had been implanted and in the second group, an aspherical IOL (Tecnis Z9000, AMO). The optical performances of the 2 IOLs were analyzed with ray-tracing software. The overall MTF of an electro-optical system and the polychromatic MTF were evaluated. RESULTS: The questionnaire analysis, confirmed with ray-tracing analysis, showed that patients with an aspherical IOL do not present with all the expected advantages. Even if there are differences in the monochromatic MTF curve computation, there is not a significant difference in the results obtained in the polychromatic MTF computation. CONCLUSION: The aspherical IOL anterior surface resolved the spherical aberration problem, but the longitudinal chromatic aberration must still be resolved.

Global gene expression in Escherichia coli K-12 during short-term and long-term adaptation to glucose-limited continuous culture conditions.

Microarray technology was used to study the cellular events that take place at the transcription level during short-term (physiological) and long-term (genetic) adaptation of the faecal indicator bacterium Escherichia coli K-12 to slow growth under limited nutrient supply. Short-term and long-term adaptation were assessed by comparing the mRNA levels isolated after 40 or 500 h of glucose-limited continuous culture at a dilution rate of 0.3 h(-1) with those from batch culture with glucose excess. A large number of genes encoding periplasmic binding proteins were upregulated, indicating that the cells are prepared for high-affinity uptake of all types of carbon sources during glucose-limited growth in continuous culture. All the genes belonging to the maltose (mal/lamB) and galactose (mgl/gal) operons were upregulated. A similar transcription pattern was observed for long-term cultures except that the expression factors were lower than in the short-term adaptation. The patterns of upregulation were confirmed by real-time RT-PCR. A switch from a fully operational citric acid cycle to the PEP-glyoxylate cycle was clearly observed in cells grown in glucose-limited continuous culture when compared to batch-grown cells and this was confirmed by transcriptome analysis. This transcriptome analysis confirms and extends the observations from previous proteome and catabolome studies in the authors' laboratory.

Analysis of stray-light effects related to intraocular lens edge design.

PURPOSE: To study the behavior of light rays that impact the frosted, double-square edge of an intraocular lens (IOL). SETTING: Optical Phisiophatological Department, University of Florence, Florence, Italy. METHODS: The interaction of light rays as a function of the edge design of a frosted, sharp-edged IOL was evaluated using an experimental eye model and an advanced ray-tracing program. The model simulates the irregularities of the frosted edge by the random overlap of sinusoidal phase gratings that have a random phase, an orientation, and a fixed period. RESULTS: The reduction in the maximum intensity of the ghost image by frosting the square edge was linked to the period of the microroughness. At a maximum microroughness, when the period is less than 5 microm, the reduction in the value of an unfrosted, square-edged IOL was 17.5%. CONCLUSIONS: The frosted edge had a small impact on reducing the intensity of the ghost image caused by the reflection of light from the double square edge of the IOL. Furthermore, in optics, the stray light from microroughness is generally considered to be a drawback because it can decrease contrast sensitivity.

Characterization of two alkane hydroxylase genes from the marine hydrocarbonoclastic bacterium Alcanivorax borkumensis.

The marine gamma-Proteobacterium Alcanivorax borkumensis is highly specialized in the assimilation of aliphatic hydrocarbons, and makes up a large part of the biomass in oil-polluted marine environments. In addition to the previously identified alkane hydroxylase AlkB1, a second alkane hydroxylase (AlkB2) showing 65% identity to the Pseudomonas aeruginosa AlkB2 alkane hydroxylase was identified. Unlike alkB1, alkB2 is not flanked by genes involved in alkane metabolism. Heterologous expression of the A. borkumensis AP1 alkB1 and alkB2 genes showed that they encode functional alkane hydroxylases with substrate ranges similar to those of their P. putida and P. aeruginosa homologues. The transcription initiation sites and levels of the alkB1, alkB2 and alkS mRNA transcripts were determined. Expression of both alkB1 and alkB2 was induced by alkanes, but transcripts corresponding to alkB1 were much more abundant than those of alkB2. An inverted repeat similar to the binding site for the P. putida GPo1 transcriptional activator AlkS was present upstream of the promoters for alkB1 and alkB2, although that of alkB2 was less well conserved, and only the transcriptional fusion of promoter PalkB1 to the reporter gene lacZ efficiently responded to n-octane. Contrary to what has been found for the P. putida GPo1 alkane degradation pathway, expression of the A. borkumensis AP1 alkS gene was not induced by alkanes, and an AlkS binding site was not present upstream of the promoter for alkS. This indicates that, in spite of the clear similarities, the A. borkumensis alk-genes are regulated by a strategy different from that of the P. putida GPo1 alk genes.

Computerized analysis of the effects of intraocular lens edge design on the quality of vision in pseudophakic patients.

PURPOSE: To study the distribution and intensity of reflected glare images from 3 intraocular lens (IOL) edge designs. SETTING: Eye Institute, University of Florence, Florence, Italy. METHODS: The interaction of light rays as a function of edge design was evaluated using an experimental eye model and an advanced ray-tracing program. Three hydrophobic acrylic IOLs were studied: an acrylic square-edged (sharp) design, an acrylic round-edged design, and a new acrylic IOL with a rounded anterior edge and a sharp posterior edge. RESULTS: The rays hitting the sharp edge of an IOL caused peripheral arcs or circles of light on the side of the retina opposite the focused image. Edge reflections also occurred with the round-edged IOL, but the intensity of the reflected image was one tenth that of the intensity with the sharp-edged design. The new round-/sharp-edged design reduced the maximum intensity of the reflected image to one third the intensity of the sharp-edged reflected image. CONCLUSIONS: The experimental findings with an acrylic IOL that combines a sharp posterior edge with a low incidence of reflected edge glare are promising.

Analysis of Pseudomonas putida alkane-degradation gene clusters and flanking insertion sequences: evolution and regulation of the alk genes.

The Pseudomonas putida GPo1 (commonly known as Pseudomonas oleovorans GPo1) alkBFGHJKL and alkST gene clusters, which encode proteins involved in the conversion of n-alkanes to fatty acids, are located end to end on the OCT plasmid, separated by 9.7 kb of DNA. This DNA segment encodes, amongst others, a methyl-accepting transducer protein (AlkN) that may be involved in chemotaxis to alkanes. In P. putida P1, the alkBFGHJKL and alkST gene clusters are flanked by almost identical copies of the insertion sequence ISPpu4, constituting a class 1 transposon. Other insertion sequences flank and interrupt the alk genes in both strains. Apart from the coding regions of the GPo1 and P1 alk genes (80-92% sequence identity), only the alkB and alkS promoter regions are conserved. Competition experiments suggest that highly conserved inverted repeats in the alkB and alkS promoter regions bind ALKS: