Quantitative determination of besifloxacin, a novel fluoroquinolone antimicrobial agent, in human tears by liquid chromatography-tandem mass spectrometry

A rapid and sensitive method was developed using high-performance liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) for the quantification of besifloxacin in human tears using sparfloxacin as the internal standard (IS). Besifloxacin was extracted from human tear samples using an ammonium formate buffer at pH 3.25. The method was validated over a concentration range of 2-2000 ng/mL, with a total run time of less than 4 min. The overall intra- and inter-day precision for this method was less than 6%. The method was used to measure besifloxacin concentrations in tear samples collected after topical ocular administration to humans; besifloxacin concentrations were 610+/-540 microg/g (15 min) and 1.60+/-2.28 microg/g (24h).     

Regulation of the incorporation of tissue factor into microparticles by serine phosphorylation of the cytoplasmic domain of tissue factor

The mechanisms that regulate the incorporation and release of tissue factors (TFs) into cell-derived microparticles are as yet unidentified. In this study, we have explored the regulation of TF release into microparticles by the phosphorylation of serine residues within the cytoplasmic domain of TF. Wild-type and mutant forms of TF, containing alanine and aspartate substitutions at Ser253 and Ser258, were overexpressed in coronary artery and dermal microvascular endothelial cells and microparticle release stimulated with PAR2 agonist peptide (PAR2-AP). The release of TF antigen and activity was then monitored. In addition, the phosphorylation state of the two serine residues within the released microparticles and the cells was monitored for 150 min. The release of wild-type TF as procoagulant microparticles peaked at 90 min and declined thereafter in both cell types. The TF within these microparticles was phosphorylated at Ser253 but not at Ser258. Aspartate substitution of Ser253 resulted in rapid release of TF antigen but not activity, whereas TF release was reduced and delayed by alanine substitution of Ser253 or aspartate substitution of Ser258. Alanine substitution of Ser258 prolonged the release of TF following PAR2-AP activation. The release of TF was concurrent with phosphorylation of Ser253 and was followed by dephosphorylation at 120 min and phosphorylation of Ser258. We propose a sequential mechanism in which the phosphorylation of Ser253 through PAR2 activation results in the incorporation of TF into microparticles, simultaneously inducing Ser258 phosphorylation. Phosphorylation of Ser258 in turn promotes the dephosphorylation of Ser253 and suppresses the release of TF.     

Partial rescue of Rett syndrome by ω-3 polyunsaturated fatty acids (PUFAs) oil

Evidence of enhanced oxidative stress (O.S.) and lipid peroxidation has been reported in patients with Rett syndrome (RTT), a relatively rare neurodevelopmental disorder progressing in 4-stages, and mainly caused by loss-of-function mutations in the methyl-CpG-binding protein 2. No effective therapy for preventing or arresting the neurologic regression in the disease in its various clinical presentations is available. Based on our prior evidence of enhanced O.S. and lipid peroxidation in RTT patients, herein we tested the possible therapeutic effects of ω-3 polyunsaturated fatty acids (ω-3 PUFAs), known antioxidants with multiple effects, on the clinical symptoms and O.S. biomarkers in the earliest stage of RTT. A total of 20 patients in stage I were randomized (n = 10 subjects per arm) to either oral supplementation with ω-3 PUFAs-containing fish oil (DHA: 72.9 ± 8.1 mg/kg b.w./day; EPA: 117.1 ± 13.1 mg/kg b.w./day; total ω-3 PUFAs: 246.0 ± 27.5 mg/kg b.w./day) for 6 months or no treatment. Primary outcomes were potential changes in clinical symptoms, with secondary outcomes including variations for five O.S. markers in plasma and/or erythrocytes (nonprotein bound iron, F₂-dihomo-isoprostanes, F₃-isoprostanes, F₄-neuroprostanes, and F₂-isoprostanes). A significant reduction in the clinical severity (in particular, motor-related signs, nonverbal communication deficits, and breathing abnormalities) together with a significant decrease in all the examined O.S. markers was observed in the ω-3 PUFAs supplemented patients, whereas no significant changes were evidenced in the untreated group. For the first time, these findings strongly suggest that a dietary intervention in this genetic disease at an early stage of its natural history can lead to a partial clinical and biochemical rescue.

Electronic supplementary material

The online version of this article (doi:10.1007/s12263-012-0285-7) contains supplementary material, which is available to authorized users.     

Dose-dependent effects of DDAVP on memory in healthy young adult males: a preliminary study

Although several studies have demonstrated the efficacy of the vasopressin analog DDAVP in enhancing human memory, no previous study has reported the dose-response relationship of DDAVP to memory in healthy young adults. The present study was undertaken to explore the dose-response curve for DDAVP on recall of implicational sentences. Five doses of DDAVP (0, 5, 15, 30, and 60 micrograms) were administered intranasally to healthy young adult male volunteers. Results demonstrated a facilitation in cued recall after treatment with the 60-micrograms dose and a general impairment in recall after treatment with the 15-micrograms dose. These effects were independent of subject's weight, vocabulary ability, and concentration of salivary cortisol.     

High-pressure effects on horse heart metmyoglobin studied by small-angle neutron scattering.

Small-angle neutron scattering experiments were performed on horse azidometmyoglobin (MbN3) at pressures up to 300 MPa. Other spectroscopic techniques have shown that a reorganization of the secondary structure and of the active site occur in this pressure range. The present measurements, performed using various concentrations of MbN3, show that the compactness of the protein is not altered as the value of its radius of gyration remains constant up to 300 MPa. The value of the second virial coefficient of the protein solution indicates that the interactions between the molecules are always strongly repulsive even if their magnitude decreases with increasing pressure. Taking advantage of the pressure-induced contrast variation, these experiments allow the partial specific volume of MbN3 to be determined as a function of pressure. Its value decreases by 5.4% between atmospheric pressure and 300 MPa. In this pressure range the isothermal compressibility of hydrated MbN3 is found to be almost constant. Its value is (1.6 +/- 0.1) 10-4 MPa-1.     

Adsorption of enteroviruses to soil cores and their subsequent elution by artificial rainwater.

The adsorption and elution of a variety of human enteroviruses in a highly permeable, sandy soil was studied by using cores (43 by 125 mm) collected from an operating recharge basin on Long Island. Viruses studied included field and reference strains of polioviruses types 1 and 3 and reference strains of coxsackie virus B3 and echovirus types 1 and 6. Viruses suspended in treated sewage effluent were allowed to percolate through soil cores, and the filtrate was assayed for unadsorbed viruses. To determine the likelihood of desorption and mobilization, soil-bound viruses were subjected to a rinse with either treated sewage effluent or simulated rainwater which reflected the anion, cation, and pH characteristics of a typical northeastern United States rainfall. The results demonstrated that all polioviruses tested, including both reference and field strains, adsorbed extremely well to cores. Adsorption was somewhat reduced when clean, unconditioned soils were used. Soil-bound poliovirus strain LSc was not significantly mobilized by flooding columns with either a sewage effluent or rainwater rinse. One virus was mobilized by both types of rinses. The amount of viruses mobilized by rainwater rinses ranged from 24 to 66%. Variable adsorption-elution results were observed with other enteroviruses. Two guanidine-resistant mutants of poliovirus LSc demonstrated a soil adsorption-elution profile different from that of the parent strain. The data support the conclusion that soil adsorption-elution behavior is strain dependent and that poliovirus, particularly strain LSc, represents an inappropriate model.     

Analysis of the cytosolic domains of the hepatitis B virus envelope proteins for their function in viral particle assembly and infectivity

The hepatitis B virus (HBV) envelope proteins have the ability to assemble three types of viral particles, (i) the empty subviral particles (SVPs), (ii) the mature HBV virions, and (iii) the hepatitis delta virus (HDV) particles, in cells that are coinfected with HBV and HDV. To gain insight into the function of the HBV envelope proteins in morphogenesis of HBV or HDV virions, we have investigated subdomains of the envelope proteins that have been shown or predicted to lie at the cytosolic face of the endoplasmic reticulum membrane during synthesis, a position prone to interaction with the inner core structure. These domains, referred to here as cytosolic loops I and II (CYL-I and -II, respectively), were subjected to mutagenesis. The mutations were introduced in the three HBV envelope proteins, designated small, middle, and large (S-HBsAg, M-HBsAg, and L-HBsAg, respectively). The mutants were expressed in HuH-7 cells to evaluate their capacity for self-assembly and formation of HBV or HDV virions when HBV nucleocapsid or HDV ribonucleoprotein, respectively, was provided. We found that SVP-competent CYL-I mutations between positions 23 and 78 of the S domain were permissive to HBV or HDV virion assembly. One mutation (P29A) was permissive for synthesis of the S- and M-HBsAg but adversely affected the synthesis or stability of L-HBsAg, thereby preventing the assembly of HBV virions. Furthermore, using an in vitro infection assay based on the HepaRG cells and the HDV model, we have shown that particles coated with envelope proteins bearing CYL-I mutations were fully infectious, hence indicating the absence of an infectivity determinant in this region. Finally, we demonstrated that the tryptophan residues at positions 196, 199, and 201 in CYL-II, which were shown to exert a matrix function for assembly of HDV particles (I. Komla-Soukha and C. Sureau, J. Virol. 80:4648-4655, 2006), were dispensable for both assembly and infectivity of HBV virions.