The role of the intensive care unit in real-time surveillance of emerging pandemics: the Italian GiViTI experience.

The prompt availability of reliable epidemiological information on emerging pandemics is crucial for public health policy-makers. Early in 2013, a possible new H1N1 epidemic notified by an intensive care unit (ICU) to GiViTI, the Italian ICU network, prompted the re-activation of the real-time monitoring system developed during the 2009-2010 pandemic. Based on data from 216 ICUs, we were able to detect and monitor an outbreak of severe H1N1 infection, and to compare the situation with previous years. The timely and correct assessment of the severity of an epidemic can be obtained by investigating ICU admissions, especially when historical comparisons can be made.

High-resolution melt analysis does not reveal mutagenic risk in sexed sperm and in vitro-derived bovine embryos.

The objectives of the present work were to verify whether simultaneous exposure to Hoechst 33342 and UV irradiation during sorting by flow cytometry may induce gene point mutations in bovine sperm and to assess whether the dye incorporated in the sperm may imply a mutagenic effect during the embryonic development. To this aim, high-resolution melt analysis (HRMA) was used to discriminate variations of single nucleotides in sexed vs. non-sexed control samples. Three batches of sorted and non-sorted commercial semen of seven bulls (42 samples) were subjected to HRMA. A set of 139 genes located on all the chromosomes was selected, and 407 regions of the genome covering a total of 83 907 bases were analyzed. Thereafter, sperm of one sexed and one non-sexed batch of each bull was used in in vitro fertilization, and the derived embryos were analyzed (n = 560). One hundred and thirty-three regions of the bovine genome, located in 40 genes, were screened for a total coverage of 23 397 bases. The comparison between the frequencies of variations, with respect to the sequences deposited, observed in the sexed and non-sexed sperm (843 vs. 770) and embryos (246 vs. 212) showed no significant differences (P > 0.05), as measured by chi-square tests. It can be concluded that staining with Hoechst 33342 and exposure to UV during sorting does not lead to significant changes in the frequencies of variants in the commercial sexed semen and in embryos produced in vitro with the same treated sperm.

Simple procedures for analyzing and purifying methylene green.

Methylene green is a versatile dye that can be used in a wide range of technical applications, most of which require the dye to be pure. Because commercial lots of methylene green are known to be heterogeneous, we report a thin layer chromatographic method for checking purity. We also describe a simple and effective flash chromatographic purification procedure for subsequent purification. The identity and purity of the dye can be checked easily using UV-visible absorption spectrum measurements or by more sophisticated procedures if necessary.

Reactions of excited states of phenoxazin-3-one dyes with amino acids.

The interaction with amino acids of the excited states of the N-oxide resazurin and its deoxygenation product resorufin, has been studied in aqueous solution at pH 7.5. Steady-state and time-resolved studies show that the fluorescence is quenched by amino acids. Complexation of the dyes in the ground state with aromatic amino acids was also observed. The singlet quenching is attributed to electron transfer from the amino acids to the excited dye based on the dependence of the bimolecular rate constants with the ionization potential of quenchers. Flash photolysis experiments allowed determination of the quenching rate constants for the triplet deactivation of dyes by several amino acids, as well as the characterization of the transients formed in the process. These data show that the triplet is also deactivated by an electron transfer process. However, the deactivation of the N-oxide dye by tryptophan can be described by a hydrogen atom transfer. The protolytic dissociation constants of the dye radical ions are reported. The irradiation of rezasurin in the presence of amino acids leads to deoxygenation of the dye to give resorufin. This process involves the triplet excited state of resazurin and is efficient only in the presence of amino acids containing the -SH group.

The excited-state interaction of resazurin and resorufin with amines in aqueous solutions. Photophysics and photochemical reactions.

The photophysics and photochemical behavior of the phenoxazin-3-one dyes, resazurin and resorufin, have been studied in aqueous solutions. The irradiation of resazurin in the presence of amines leads to deoxygenation of the N-oxide group, giving resorufin. This photoreaction is highly dependent on the amine structure and is efficient only in the presence of tertiary aliphatic amines. The absorption and fluorescence properties of these dyes are dependent on pH. At pH above 7.5 both dyes are in their anionic form. For resorufin this form is highly fluorescent (phiF = 0.75). At lower pH the fluorescence is strongly reduced. The N-oxide dye presents a very weak fluorescence quantum yield (0.11), which also is reduced at low pH. Flash photolysis experiments allowed characterization of the triplet state and the transients formed after irradiation of these dyes in the absence and presence of amines. The triplet quantum yields are 0.08 for resazurin and 0.04 for resorufin. The photodeoxygenation of N-oxide in the presence of amines occurs from the triplet state.

Fluorescence quenching of anthracene by indole derivatives in phospholipid bilayers.

The quenching of anthracene fluorescence by indole, 1,2-dimethylindole (DMI), tryptophan (Trp) and indole 3-acetic acid (IAA) in palmitoyloleoylphosphatidylcholine (POPC) lipid bilayers was investigated. A very efficient quenching of the anthracene fluorescence in the lipid membrane is observed. Stern-Volmer plots are linear for DMI but present a downward curvature for the other quenchers. This was interpreted as an indication of the presence of an inaccessible fraction of anthracene molecules. By a modified Stern-Volmer analysis the fraction accessible to the quenchers and the quenching constant were determined. The changes in the fluorescence emission spectrum of indole and DMI have been used to calculate the partition constants of these probes into the membranes, and bimolecular quenching rate constants were determined in terms of the local concentration of quencher in the lipid bilayer. The rate constants are lower than those in homogeneous solvents, which may be ascribed to a higher viscosity of the bilayer. No changes in the emission spectra of Trp and IAA are observed in the presence of vesicles, indicating that these probes locate preferentially in the aqueous phase, or in close proximity to the vesicular external interface in a medium resembling pure water. In these cases quenching rate constants were determined in terms of the analytical concentration. In the quenching by DMI a new, red shifted, emission band appears; it is similar to that observed in non-polar solvents and it is ascribable to an exciplex emission. The exciplex band is absent in the quenching by IAA and Trp and only very weakly present when the quencher is indole. From the position of the maximum of the exciplex emission, a relatively high local polarity could be estimated for the region of the bilayer where the quenching reaction takes place.

Exciplex-type behavior and partition of 3-substituted indole derivatives in reverse micelles made with benzylhexadecyldimethylammonium chloride, water and benzene.

The fluorescence properties of 3-methylindole (MI), 3-indoleacetic acid (IAA), 3-indoleethyltrimethylammonium bromide (IETA), L-tryptophan (Trp) and tryptamine hydrochloride (TA) were studied in reverse micelles solutions made with the cationic surfactant benzylhexadecyldimethylammonium chloride (BHDC) in benzene as a function of the molar ratio water/surfactant R (= [H2O]/[BHDC]). The fluorescence quenching of the model compound MI by benzene in cyclohexane solutions and by BHDC in benzene solutions were also studied in detail. The fluorescence of MI in benzene is characteristic of a charge-transfer exciplex. The exciplex is quenched by the presence of BHDC, due to the interactions of the surfactant ion pairs with the polar exciplex. In reverse micelle solutions at low R values, all the indoles show exciplex-type fluorescence. As R increases, the fluorescence behavior strongly depends on the nature of the indole derivative. The anionic IAA remains anchored to the cationic interface and its fluorescence is quenched upon water addition due to the increases of interface's micropolarity. For IETA, TA and Trp an initial fluorescence quenching is observed at increasing R, but a fluorescence recovery is observed at R > 5, indicating a probe partition between the micellar interface and the water pool. For the neutral MI, the fluorescence changes with R indicate the partition of the probe between the micellar interface and the bulk benzene pseudophase. A simple two-site model is proposed for the calculation of the partition constants K as a function of R. In all cases, the calculation showed that even at the highest R value, about 90% of the indole molecules remain associated at the micellar interface.

The photoprotector mechanism of mycosporine-like amino acids. Excited-state properties and photostability of porphyra-334 in aqueous solution.

The photostability and photophysical parameters of an aqueous solution of the mycosporine-like amino acid (MAA) porphyra-334 have been determined. The excited-singlet state lifetime, measured by time-correlated single photon counting, was 0.4 ns. Laser flash photolysis experiments at 355 nm did not show any transient species. The triplet state of porphyra-334 was sensitized by triplet-triplet energy transfer. The T-T absorption spectrum was determined and the maximal absorption coefficient at 440 nm was estimated to be 1 x 10(4) M(-1) cm(-1). In this way an upper limit for the intersystem crossing quantum yield was determined. The very low quantum yield of fluorescence (phiF = 0.0016) and triplet formation (phiT < 0.05) together with a photodecomposition quantum yield of 2-4 x 10(-4), in the absence and the presence of oxygen respectively, can be explained by a very fast internal conversion process. These results support the photoprotective role assigned to this MAA in living systems.

Andrological study of two young identical twin bulls.

A study was conducted on two young Italian Holstein bulls obtained by embryonic splitting and, therefore, genetically identical. The andrological evaluation of the subjects revealed a significant difference in semen freezability, valued according to forward motility after thawing (57.34 vs. 46.92; P < 0.01). There was, however, no difference in the quality of fresh semen.