From bench to bedside: in vitro and in vivo evaluation of a neonate-focused nebulized surfactant delivery strategy.

BACKGROUND: Non-invasive delivery of nebulized surfactant has been a neonatology long-pursued goal. Nevertheless, the clinical efficacy of nebulized surfactant remains inconclusive, in part, due to the great technical challenges of depositing nebulized drugs in the lungs of preterm infants. The aim of this study was to investigate the feasibility of delivering nebulized surfactant (poractant alfa) in vitro and in vivo with an adapted, neonate-tailored aerosol delivery strategy. METHODS: Particle size distribution of undiluted poractant alfa aerosols generated by a customized eFlow-Neos nebulizer system was determined by laser diffraction. The theoretical nebulized surfactant lung dose was estimated in vitro in a clinical setting replica including a neonatal continuous positive airway pressure (CPAP) circuit, a cast of the upper airways of a preterm neonate, and a breath simulator programmed with the tidal breathing pattern of an infant with mild respiratory distress syndrome (RDS). A dose-response study with nebulized surfactant covering the 100-600 mg/kg nominal dose-range was conducted in RDS-modelling, lung-lavaged spontaneously-breathing rabbits managed with nasal CPAP. The effects of nebulized poractant alfa on arterial gas exchange and lung mechanics were assessed. Exogenous alveolar disaturated-phosphatidylcholine (DSPC) in the lungs was measured as a proxy of surfactant deposition efficacy. RESULTS: Laser diffraction studies demonstrated suitable aerosol characteristics for inhalation (mass median diameter, MMD = 3 µm). The mean surfactant lung dose determined in vitro was 13.7% ± 4.0 of the 200 mg/kg nominal dose. Nebulized surfactant delivered to spontaneously-breathing rabbits during nasal CPAP significantly improved arterial oxygenation compared to animals receiving CPAP only. Particularly, the groups of animals treated with 200 mg/kg and 400 mg/kg of nebulized poractant alfa achieved an equivalent pulmonary response in terms of oxygenation and lung mechanics as the group of animals treated with instilled surfactant (200 mg/kg). CONCLUSIONS: The customized eFlow-Neos vibrating-membrane nebulizer system efficiently generated respirable aerosols of undiluted poractant alfa. Nebulized surfactant delivered at doses of 200 mg/kg and 400 mg/kg elicited a pulmonary response equivalent to that observed after treatment with an intratracheal surfactant bolus of 200 mg/kg. This bench-characterized nebulized surfactant delivery strategy is now under evaluation in Phase II clinical trial (EUDRACT No.:2016-004547-36).

Top-down sequencing of Apis dorsata apamin by MALDI-TOF MS and evidence of its inactivity against microorganisms.

Apis mellifera venom is one of the best characterized venoms among Hymenoptera, yet relatively little is known about venom belonging to other species in the genus Apis. Melittin, one of the most important bioactive peptides, has been isolated and characterized in A. mellifera, Apis cerana, Apis dorsata and Apis florea, while apamin has been only characterized in A. mellifera and A. cerana. At present, no information is available about the sequence of A. dorsata apamin. Moreover, while the antiseptic properties of melittin and MCD peptides are well documented, the antimicrobial activity of apamin has never been tested. In the present study, we isolated and characterized apamin from the venom of the giant honeybee A. dorsata. We tested the activity of apamin against bacteria and yeasts in a microbiological assay to gain a more complete understanding of the antimicrobial competence of the medium molecular weight venom fraction. We show that A. dorsata apamin toxin has the same primary sequence as apamin in A. mellifera and A. cerana, yet with a different C-terminal amidation. We did not find any antiseptic activity of apamin against any of the tested microorganisms. We discuss the evolutionary processes connected to the ecological context of venom use that drove the generation of Apis venom complexity.

Preliminary evaluation of a lateral flow immunoassay device for screening urine samples for the presence of sulphamethazine.

A lateral flow immunoassay (LFIA) device was developed and applied to testing urine samples for residues of the antimicrobial sulphamethazine (SMZ). This report describes the preparation of a rat monoclonal antibody to SMZ and its characterisation in an ELISA format. Apart from SMZ, the antibody showed high (> or =50%) cross-reactivity to N4-acetyl-sulphamethazine (55%), sulphamerazine (59%) and sulphisoxazole (50%) and lower cross-reactivity of 18% to sulphachlorpyridazine and sulphadiazine. The LFIA device consisted of a nitrocellulose membrane spotted with SMZ-ovalbumin and goat anti-mouse antibody as capture line and control line, respectively. Mouse anti-rat IgG F(ab')2 fragment specific antibody, adsorbed to colloidal carbon, was used as the detection ligand in the LFIA. The LFIA device had a cut-off value of 6.3 ng/ml in diluted (1/10) urine. Urine samples from SMZ-treated pigs, and bovine and porcine urine samples fortified with SMZ were used for a blind, four-laboratory evaluation of the performance of the LFIA device. Concentrations of SMZ in the test samples (n=29), as determined by LC-MS/MS, ranged from 0 (<3) to 1174 ng/ml. The evaluation of the LFIA device showed an overall sensitivity of 100%, a specificity of 71%, and positive and negative prediction values of 73% and 100%, respectively. The LFIA device has been fabricated as a test kit for determining SMZ residues in animals produced for slaughter.

First search for the flavor changing neutral current decay D0-->gammagamma.

Using 13.8 fb(-1) of data collected at or just below the Upsilon(4S) with the CLEO detector, we report the result of a search for the flavor changing neutral current process D0-->gammagamma. We observe no significant signal for this decay mode and determine 90% confidence level upper limits on the branching fractions B(D0-->gammagamma)/B(D0-->pi(0)pi(0))<0.033 and B(D0-->gammagamma)<2.9 x 10(-5).

Metabolic responses of the limpet Patella caerulea (L.) to anoxia and dehydration.

This study examined the metabolic responses of the limpet Patella caerulea (L.) to anoxia and dehydration, attempting to tease apart the effect of these two stressful conditions, which are often not clearly distinguished in experiments. Specimens were exposed to: (a) oxygen-free sea water; (b) oxygen-saturated water (controls); (c) low-humidity air (55% RH); and (d) high-humidity air (100% RH). For each of the treatments, we took samples of five specimens after 6 and 18 h of exposure to the experimental conditions and determined the concentrations in the foot muscle of succinate, acetate, propionate, aspartate and alanine. Exposure to anoxia caused an increase in the levels of succinate (6 and 18 h) and acetate and propionate (18 h) with respect to control specimens. Anoxia also induced a decrease of aspartate and an increase of alanine after both 6 and 18 h. Exposure to both moist and dry air generally had negligible effects on the organic acid levels. Aspartate content increased after 18 h of exposure to moist air. Alanine levels also increased with respect to control values after exposure to air, with dry air having the more pronounced effect. In conclusion, the results of this study suggest that one should be cautious when inferring anaerobic conditions from the simple exposure of intertidal species to air, without strict control of the experimental conditions and actual respiration rates.

Defensive role of secretion of ectal mandibular glands of the wasp Polistes dominulus.

Ectal mandibular glands of insects are exocrine glands localized in the anterior-lateral region of the head, close to the base of the mandibles. In social wasps, the gland is composed of secretory cells and a reservoir into which the secretion accumulates. At the time of emission the secretion flows onto a specialized cuticular area on the outside of the base of the mandibles. Secretion of Polistes dominulus is emitted only when wasps are greatly disturbed or in the presence of predators, and its function seems to be mainly defensive. Morphometric studies did not reveal any size differences between the glands of the queens and those of the workers. GC-MS analyses of the glands identified 32 compounds, mainly acids and aldehydes in the range C2-C18. The overall odor, caused by the mixture of aldehydes, is distinct. Workers do not respond strongly to the odor. The secretion probably serves as a warning signal to vertebrate predators.

Nestmate recognition in Parischnogaster striatula (Hymenoptera Stenogastrinae), visual and olfactory recognition cues.

The recognition of nestmates from alien individuals is a well known phenomenon in social insects. In the stenogastrine wasp Parischnogaster striatula, we investigated the ability of females to recognize nestmates and the cues on which such recognition is based. Recognition of nestmates was observed in naturally occurring interactions between wasps approaching a nest and the resident females on that nest. This recognition was confirmed in experiments in which nestmates or alien conspecifics were presented to resident females. In naturally occurring interactions, nestmates generally approach their nest with a direct flight, while aliens usually hover in front of the nest before landing. In experiments in which the presented wasps were placed close to the nest in a direct manner, antennation of the presented wasp generally occurred, indicating that chemical cues are involved. Experiments in which dead alien individuals, previously washed in hexane, and then reapplied with extracts were recognized by colonies giving further evidence that chemical cues mediate nestmate recognition. Epicuticular lipids, known to be nestmate recognition cues in social insects, were chemically analysed by GC-MS for 44 P. striatula females from two different populations (13 different colonies). Discriminant analysis was performed on the data for the lipid mixture composition. The discriminant model showed that, in the samples from these two populations, 68.2% and 81.9% of the specimens could be correctly assigned to their colony.

Bounds on the CP asymmetry in b --> sgamma decays.

We have measured the CP asymmetry A(CP) identical with[gamma(b-->sgamma)-gammab-->sgamma)]/[gamma(b-->sgamma)+gamma(b-->sgamma)] to be A(CP) = (-0.079+/-0.108+/-0.022) (1.0+/-0.030), implying that, at 90% confidence level, A(CP) lies between -0.27 and +0.10. These limits rule out some extreme non-standard-model predictions, but are consistent with most, as well as with the standard model.

Study of tau decays to six pions and a neutrino.

The tau decays to six-pion final states have been studied with the CLEO detector at the Cornell Electron Storage Ring. The measured branching fractions are B(tau(-)-->2pi(-)pi(+)3pi(0)nu(tau)) = (2.2+/-0.3+/-0.4)x10(-4) and B(tau(-)-->3pi(-)2pi(+)pi(0)nu(tau)) = (1.7+/-0.2+/-0.2)x10(-4). A search for substructure in these decays shows that they are saturated by intermediate states with eta or omega mesons. We present the first observation of the decay tau(-)-->2pi(-)pi(+)omega(nu)tau and the branching fraction is measured to be (1.2+/-0.2+/-0.1)x10(-4). The measured branching fractions are in good agreement with the isospin expectations but somewhat below the conserved-vector-current predictions.

Observation of new states decaying into Lambda+(c)pi(-)pi(+).

Using 13.7 fb(-1) of data recorded by the CLEO detector at the Cornell Electron Storage Ring, we investigate the spectrum of charmed baryons which decay into Lambda+(c)pi(-)pi(+) and are more massive than the Lambda+(c)(2625) baryon. We find evidence for two new states: one is broad and has an invariant mass roughly 480 MeV above that of the Lambda+(c) baryon; the other is narrow with an invariant mass of 596+/-1+/-2 MeV above the Lambda+(c) mass.

Observation of the Omega(0)(c) Charmed Baryon at CLEO.

The CLEO experiment at the CESR collider has used 13.7 fb(-1) of data to search for the production of the Omega(0)(c) (css ground state) in e(+)e(-) collisions at square root of (s) approximately 10.6 GeV. The modes used to study the Omega(0)(c) are Omega(-)pi(+), Omega(-)pi(+)pi(0), Xi-K-pi(+)pi(+), Xi0K-pi(+), and Omega(-)pi(+)pi(+)pi(-). We observe a signal of 40.4+/-9.0(stat) events at a mass of 2694.6+/-2.6(stat)+/-1.9(syst) MeV/c(2), for all modes combined.

Determination of the levels of aromatic amines in indoor and outdoor air in Italy.

We studied the concentration of 10 primary aromatic amines (AA), which are classified as suspected carcinogens, in indoor and outdoor air in Italy. The measured AA included: aniline, o-toluidine, m-toluidine, p-toluidine, 2,3-dimethylaniline, 2,4-dimethylaniline, 2,5-dimethylaniline, 2,6-dimethylaniline, 2-naphtylamine and 4-aminobiphenyl. In the indoor environment (homes, offices and public buildings) the level of contamination (expressed as sum of 9 AA, excluding aniline) varied from 3 ng/m3 (hospital ward) to 207 ng/m3 (discotheque). In most indoor environments with no contamination from cigarette smoke the AA levels were below 20 ng/m3, whereas in the presence of smokers higher values were observed. Aniline levels were more erratic (varying from 53 ng/m3 (office of non-smokers) to 1929 ng/m3 (discotheque) and were not related to cigarette smoke. The concentration range of AA (excluding aniline) in the outside air varied from 3 ng/m3 (Siena) to 104 ng/m3 (Brindisi); aniline concentration was extremely variable. Most samples of outdoor air had AA levels lower than 40 ng/m3. In conclusion, AA are widespread air contaminants and attain a high concentration in heavily contaminated indoor environments, due to smoking and poor ventilation. AA occasionally attain a high level in outdoor air as well. Therefore, a strategy of reduction of the exposure to AA should consider the abatement of multiple sources of contamination.

Observation of B --> K(+/-) pi(0) and B --> (K)0 pi(0), and evidence for B --> pi(+)pi(-).

We have studied charmless hadronic decays of B mesons into two-body final states with kaons and pions and observe three new processes with the following branching fractions: beta(B-->pi(+)pi(-)) = (4.3(+1. 6)(-1.4)+/-0.5)x10(-6), beta(B-->K(0)pi(0)) = (14.6(+5.9+2.4)(-5.1-3. 3))x10(-6), and beta(B-->K(+)/-pi(0)) = (11.6(+3.0+1.4)(-2.7-1.3))x10(-6). We also update our previous measurements for the decays B-->K(+)/-pi(-/+) and B+/--->K(0)pi(+/-).

Two-body B meson decays to eta and eta('): observation of B --> eta K*.

In a sample of 19 x 10(6) produced B mesons, we have observed the decays B-->eta K(*) and improved our previous measurements of B-->eta'K. The branching fractions we measure for these decay modes are B(B+-->eta K(*+)) = (26.4(+9.6)(-8.2)+/-3.3)x10(-6), B(B(0)-->eta K(*0)) = (13.8(+5.5)(-4.6)+/-1.6)x10(-6), B(B(+)-->eta'K(+) = (80(+10)(-9)+/-7)x10(-6), and B(B(0)-->eta'K0) = (89(+18)(-16)+/-9)x10(-6). We have searched with comparable sensitivity for related decays and report upper limits for these branching fractions.

Measurement of charge asymmetries in charmless hadronic B meson decays.

We search for CP-violating charge asymmetries (alpha(CP)) in the B meson decays to K(+/-)pi(-/+), K(+/-)pi(0), K(0)(S)pi(+/-), K(+/-)eta('), and omega pi(+/-). Using 9.66 million upsilon(4S) decays collected with the CLEO detector, the statistical precision on alpha(CP) is in the range of +/-0.12 to +/-0.25 depending on decay mode. While CP-violating asymmetries of up to +/-0.5 are possible within the standard model, the measured asymmetries are consistent with zero in all five decay modes studied.

Study of charmless hadronic B meson decays to pseudoscalar-vector final states.

We report results of searches for charmless hadronic B meson decays to pseudoscalar( pi(+/-), K+/-, pi(0), or K(0)(S))-vector( rho, K(*), or omega) final states. By using 9.7x10(6) BB pairs collected with the CLEO detector, we report the first observation of B(-)--->pi(-)rho(0), B(0)-->pi(+/-)rho(-/+), and B(-)-->pi(-)omega, which are expected to be dominated by hadronic b-->u transitions. The measured branching fractions are (10.4(+3.3)(-3.4)+/-2.1)x10(-6), (27.6(+8.4)(-7.4)+/-4.2)x10(-6), and (11.3(+3.3)(-2.9)+/-1. 4)x10(-6), respectively. Branching fraction upper limits are set for all of the other decay modes investigated.

Liquid chromatography tandem mass spectrometric quantitation of sulfamethazine and its metabolites: direct analysis of swine urine by triple quadrupole and by ion trap mass spectrometry.

This work describes a new method for the quantitation of trace amounts of sulfamethazine (SMZ) and its main metabolite, N4-acetylsulfamethazine (Ac-SMZ), in swine urine, using high-performance liquid chromatography (HPLC) tandem mass spectrometric analysis of crude urine after addition of internal standard and simple dilution with water. The aim was to determine whether residues of this sulfamidic drug, normally administered to swine in order to prevent infectious diseases, were present in urine at levels lower than those permitted by regulatory authorities before human consumption (EU Project SMT, contract number CT 96-2092). A 10 microL volume of diluted urine was injected into a very short, narrow-bore chromatographic column (Zorbax SB-C18 2.1 i. d. x30 mm length, 3.5 microm pore size). Elution of the analytes of interest was achieved in less than seven minutes using a rapid gradient (from 20 to 80% methanol in 3 minutes). Either a PE Sciex API 365 triple quadrupole (QqQ), operated in the selected reaction monitoring (SRM) mode, or a Finnigan LCQ ion trap (IT) mass spectrometer, operated in narrow-range product ion scan, was used as the final detector. Electrospray (ESI) was used as the ionization technique. A comparison of the two tandem mass spectrometers was performed by analyzing the same set of test samples, at three concentration levels, on three different days. Linearity of responses of the calibration standards, intra- and inter-assay precision of the samples, specificity and limits of detection were evaluated for both systems. Both the QqQ and the IT instrument was suitable for rapid, sensitive and specific determination of the analytes, although the overall performance of the QqQ was slightly superior in terms of linearity, precision and sensitivity.

Use of Dufour's gland secretion in nest defence and brood nutrition by hover wasps (Hymenoptera, Stenogastrinae).

Social wasps of the subfamily Stenogastrinae produce an abdominal secretion that is used in two distinct biological contexts. First, the secretion plays an important role in larval nutrition where it serves as a substrate in which food is placed by the adults for eventual consumption by the larvae. Second, in several species, females apply the same secretion to the substrate on which their nests are constructed, where it constitutes a sticky barrier that defends the immature brood from predation by ants. This paper describes for the first time ant guard construction behaviour of three species of stenogastrine wasps belonging to the genera Eustenogaster and Liostenogaster. The identification of compounds making up these secretions was also performed by gas chromatography-mass spectrometry. Ant guards and brood secretions were similar, with saturated and unsaturated long chain hydrocarbons and alcohols as major components. We further confirm that the glandular source of abdominal secretion is the Dufour's gland. This gland contains the same hydrocarbons, and in the same proportions as ant guards and brood secretion. We discuss the fundamental importance of Dufour's gland secretion in the social life of these wasps by comparing species with and without ant guards within the subfamily.

Use of solid-phase microextraction in the investigation of chemical communication in social wasps.

Solid-phase microextraction has been used to investigate chemical communication in several social wasp species. Using the technique to analyse exocrine gland secretions, we demonstrate that the results are comparable with those obtained with the more classical methods that use solvents, eliminating, in many cases, the shortcomings of these methods in insect pheromone analysis. As a result of its simplicity this technique is very suitable for research on the chemical ecology of social wasps, and on insect communication in general.