Novel Fluorescein Angiography-Based Computer-Aided Algorithm for Assessment of Retinal Vessel Permeability

Purpose

To present a novel method for quantitative assessment of retinal vessel permeability using a fluorescein angiography-based computer algorithm.

Methods

Twenty-one subjects (13 with diabetic retinopathy, 8 healthy volunteers) underwent fluorescein angiography (FA). Image pre-processing included removal of non-retinal and noisy images and registration to achieve spatial and temporal pixel-based analysis. Permeability was assessed for each pixel by computing intensity kinetics normalized to arterial values. A linear curve was fitted and the slope value was assigned, color-coded and displayed. The initial FA studies and the computed permeability maps were interpreted in a masked and randomized manner by three experienced ophthalmologists for statistical validation of diagnosis accuracy and efficacy.

Results

Permeability maps were successfully generated for all subjects. For healthy volunteers permeability values showed a normal distribution with a comparable range between subjects. Based on the mean cumulative histogram for the healthy population a threshold (99.5%) for pathological permeability was determined. Clear differences were found between patients and healthy subjects in the number and spatial distribution of pixels with pathological vascular leakage. The computed maps improved the discrimination between patients and healthy subjects, achieved sensitivity and specificity of 0.974 and 0.833 respectively, and significantly improved the consensus among raters for the localization of pathological regions.

Conclusion

The new algorithm allows quantification of retinal vessel permeability and provides objective, more sensitive and accurate evaluation than the present subjective clinical diagnosis. Future studies with a larger patients’ cohort and different retinal pathologies are awaited to further validate this new approach and its role in diagnosis and treatment follow-up. Successful evaluation of vasculature permeability may be used for the early diagnosis of brain microvascular pathology and potentially predict associated neurological sequelae. Finally, the algorithm could be implemented for intraoperative evaluation of micovascular integrity in other organs or during animal experiments.     

Evidence of the Presence of a Functional Dot/Icm Type IV-B Secretion System in the Fish Bacterial Pathogen Piscirickettsia salmonis

Piscirickettsia salmonis is a fish bacterial pathogen that has severely challenged the sustainability of the Chilean salmon industry since its appearance in 1989. As this Gram-negative bacterium has been poorly characterized, relevant aspects of its life cycle, virulence and pathogenesis must be identified in order to properly design prophylactic procedures. This report provides evidence of the functional presence in P. salmonis of four genes homologous to those described for Dot/Icm Type IV Secretion Systems. The Dot/Icm System, the major virulence mechanism of phylogenetically related pathogens Legionella pneumophila and Coxiella burnetii, is responsible for their intracellular survival and multiplication, conditions that may also apply to P. salmonis. Our results demonstrate that the four P. salmonis dot/icm homologues (dotB, dotA, icmK and icmE) are expressed both during in vitro tissue culture cells infection and growing in cell-free media, suggestive of their putative constitutive expression. Additionally, as it happens in other referential bacterial systems, temporal acidification of cell-free media results in over expression of all four P. salmonis genes, a well-known strategy by which SSTIV-containing bacteria inhibit phagosome-lysosome fusion to survive. These findings are very important to understand the virulence mechanisms of P. salmonis in order to design new prophylactic alternatives to control the disease.     

Crystal Structure,SAXS and Kinetic Mechanism of Hyperthermophilic ADP-Dependent Glucokinase from Thermococcus litoralis Reveal a Conserved Mechanism for Catalysis

ADP-dependent glucokinases represent a unique family of kinases that belong to the ribokinase superfamily, being present mainly in hyperthermophilic archaea. For these enzymes there is no agreement about the magnitude of the structural transitions associated with ligand binding and whether they are meaningful to the function of the enzyme. We used the ADP-dependent glucokinase from Termococcus litoralis as a model to investigate the conformational changes observed in X-ray crystallographic structures upon substrate binding and to compare them with those determined in solution in order to understand their interplay with the glucokinase function. Initial velocity studies indicate that catalysis follows a sequential ordered mechanism that correlates with the structural transitions experienced by the enzyme in solution and in the crystal state. The combined data allowed us to resolve the open-closed conformational transition that accounts for the complete reaction cycle and to identify the corresponding clusters of aminoacids residues responsible for it. These results provide molecular bases for a general mechanism conserved across the ADP-dependent kinase family.     

Circulating Endothelial Cells and Procoagulant Microparticles in Patients with Glioblastoma: Prognostic Value

Aim

Circulating endothelial cells and microparticles are prognostic factors in cancer. However, their prognostic and predictive value in patients with glioblastoma is unclear. The objective of this study was to investigate the potential prognostic value of circulating endothelial cells and microparticles in patients with newly diagnosed glioblastoma treated with standard radiotherapy and concomitant temozolomide. In addition, we have analyzed the methylation status of the MGMT promoter.

Methods

Peripheral blood samples were obtained before and at the end of the concomitant treatment. Blood samples from healthy volunteers were also obtained as controls. Endothelial cells were measured by an immunomagnetic technique and immunofluorescence microscopy. Microparticles were quantified by flow cytometry. Microparticle-mediated procoagulant activity was measured by endogen thrombin generation and by phospholipid-dependent clotting time. Methylation status of MGMT promoter was determined by multiplex ligation-dependent probe amplification.

Results

Pretreatment levels of circulating endothelial cells and microparticles were higher in patients than in controls (p<0.001). After treatment, levels of microparticles and thrombin generation decreased, and phospholipid-dependent clotting time increased significantly. A high pretreatment endothelial cell count, corresponding to the 99^th percentile in controls, was associated with poor overall survival. MGMT promoter methylation was present in 27% of tumor samples and was associated to a higher overall survival (66 weeks vs 30 weeks, p<0.004).

Conclusion

Levels of circulating endothelial cells may have prognostic value in patients with glioblastoma.     

Effective Population Size,Genetic Variation,and Their Relevance for Conservation: The Bighorn Sheep in Tiburon Island and Comparisons with Managed Artiodactyls

The amount of genetic diversity in a finite biological population mostly depends on the interactions among evolutionary forces and the effective population size (N _e) as well as the time since population establishment. Because the N _e estimation helps to explore population demographic history, and allows one to predict the behavior of genetic diversity through time, N _e is a key parameter for the genetic management of small and isolated populations. Here, we explored an N _e-based approach using a bighorn sheep population on Tiburon Island, Mexico (TI) as a model. We estimated the current (N _crnt) and ancestral stable (N _stbl) inbreeding effective population sizes as well as summary statistics to assess genetic diversity and the demographic scenarios that could explain such diversity. Then, we evaluated the feasibility of using TI as a source population for reintroduction programs. We also included data from other bighorn sheep and artiodactyl populations in the analysis to compare their inbreeding effective size estimates. The TI population showed high levels of genetic diversity with respect to other managed populations. However, our analysis suggested that TI has been under a genetic bottleneck, indicating that using individuals from this population as the only source for reintroduction could lead to a severe genetic diversity reduction. Analyses of the published data did not show a strict correlation between H _E and N _crnt estimates. Moreover, we detected that ancient anthropogenic and climatic pressures affected all studied populations. We conclude that the estimation of N _crnt and N _stbl are informative genetic diversity estimators and should be used in addition to summary statistics for conservation and population management planning.     

Synthesis of new β-amidodehydroaminobutyric acid derivatives and of new tyrosine derivatives using copper catalyzed C–N and C–O coupling reactions

Several β-amidodehydroaminobutyric acid derivatives were prepared from N,C-diprotected β-bromodehydroaminobutyric acids and amides by a copper catalyzed C–N coupling reaction. The best reaction conditions include the use of a catalytic amount of CuI, N,N′-dimethylethylenediamine as ligand and K₂CO₃ as base in toluene at 110 °C. The stereochemistry of the products was determined using NOE difference experiments and the results obtained are in agreement with an E-stereochemistry. Thus, the stereochemistry is maintained in the case of the E-isomers of β-bromodehydroaminobutyric acid derivatives, but when the Z-isomers were used as substrates the reaction proceeds with inversion of configuration. The use of β-bromodehydrodipeptides as substrates was also tested. It was found that the reaction outcome depend on the stereochemistry of the β-bromodehydrodipeptide and on the nature of the first amino acid residue. The products isolated were the β-amidodehydrodipeptide derivatives and/or the corresponding dihydropyrazines. The same catalytic system (CuI/N,N′-dimethylethylene diamine) was used in the C–O coupling reactions between a tyrosine derivative and aryl bromides. The new O-aryltyrosine derivatives were isolated in moderate to good yields. The photophysical properties of two of these compounds were studied in four solvents of different polarity. The results show that these compounds after deprotection can be used as fluorescence markers.     

Synthesis and antibacterial activity of conjugates between norfloxacin and analogues of the siderophore vanchrobactin

From synthetic functionalized analogues of vanchrobactin, a siderophore produced by the fish pathogenic bacteria Vibrio anguillarum serotype O2, several vanchrobactin analogues–norfloxacin conjugates were obtained and their antimicrobial activities against the wild-type and mutant strains of Vibrio anguillarum serotype O2 have been determined.     

Spectroscopic,computational modeling and cytotoxicity of a series of meso-phenyl and meso-thienyl-BODIPYs

A series of twenty-two BODIPY compounds were synthesized, containing various meso-phenyl and meso-thienyl groups, and their spectroscopic and structural properties were investigated using both experimental and computational methods. Further functionalization of the BODIPY framework via iodination at the 2,6-pyrrolic positions was explored in order to determine the effect of these heavy atoms on the photophysical and cytotoxicity of the meso-aryl-BODIPYs. BODIPYs bearing meso-thienyl substituents showed the largest red-shifted absorptions and emissions and reduced fluorescence quantum yields. The phototoxicity of the BODIPYs in human carcinoma HEp2 cells depends on both the presence of iodines and the nature of the meso-aryl groups. Six of the eleven 2,6-diiodo-BODIPYs investigated showed at least a sevenfold enhancement in phototoxicity (IC₅₀ = 3.5–28 μM at 1.5 J/cm²) compared with the non-iodinated BODIPYs, while the others showed no cytotoxicity, while their singlet oxygen quantum yields ranged from 0.02 to 0.76. Among the series investigated, BODIPYs 2a and 4a bearing electron-donating meso-dimethoxyphenyl substituents showed the highest phototoxicity and dark/phototoxicity ratio, and are therefore the most promising for application in PDT.