An innovative hematopoietic stem cell gene therapy approach benefits CLN1 disease in the mouse model.

Hematopoietic stem and progenitor cells (HSPCs) can establish a long-lasting microglia-like progeny in the central nervous system of properly myeloablated hosts. We exploited this approach to treat the severe CLN1 neurodegenerative disorder, which is the most aggressive form of neuronal ceroid lipofuscinoses due to palmitoyl-protein thioesterase-1 (PPT1) deficiency. We here provide the first evidence that (i) transplantation of wild-type HSPCs exerts partial but long-lasting mitigation of CLN1 symptoms; (ii) transplantation of HSPCs over-expressing hPPT1 by lentiviral gene transfer enhances the therapeutic benefit of HSPCs transplant, with first demonstration of such a dose-effect benefit for a purely neurodegenerative condition like CLN1 disease; (iii) transplantation of hPPT1 over-expressing HSPCs by a novel intracerebroventricular (ICV) approach is sufficient to transiently ameliorate CLN1-symptoms in the absence of hematopoietic tissue engraftment of the transduced cells; and (iv) combinatorial transplantation of transduced HSPCs intravenously and ICV results in a robust therapeutic benefit, particularly on symptomatic animals. Overall, these findings provide first evidence of efficacy and feasibility of this novel approach to treat CLN1 disease and possibly other neurodegenerative conditions, paving the way for its future clinical application.

Magnetic Resonance Acoustic Radiation Force Imaging (MR-ARFI) for the monitoring of High Intensity Focused Ultrasound (HIFU) ablation in anisotropic tissue.

OBJECTIVE: We introduce a non-invasive MR-Acoustic Radiation Force Imaging (ARFI)-based elastography method that provides both the local shear modulus and temperature maps for the monitoring of High Intensity Focused Ultrasound (HIFU) therapy. MATERIALS AND METHODS: To take tissue anisotropy into account, the local shear modulus µ is determined in selected radial directions around the focal spot by fitting the phase profiles to a linear viscoelastic model, including tissue-specific mechanical relaxation time τ. MR-ARFI was evaluated on a calibrated phantom, then applied to the monitoring of HIFU in a gel phantom, ex vivo and in vivo porcine muscle tissue, in parallel with MR-thermometry. RESULTS: As expected, the shear modulus polar maps reflected the isotropy of phantoms and the anisotropy of muscle. In the HIFU monitoring experiments, both the shear modulus polar map and the thermometry map were updated with every pair of MR-ARFI phase images acquired with opposite MR-ARFI-encoding. The shear modulus was found to decrease (phantom and ex vivo) or increase (in vivo) during heating, before remaining steady during the cooling phase. The mechanical relaxation time, estimated pre- and post-HIFU, was found to vary in muscle tissue. DISCUSSION: MR-ARFI allowed for monitoring of viscoelasticity changes around the HIFU focal spot even in anisotropic muscle tissue.

Monitoring MR-guided high intensity focused ultrasound therapy using transient supersonic shear wave MR-elastography.

Objective.The aim of the paper is to propose an all-in-one method based on magnetic resonance-supersonic shear wave imaging (MR-SSI) and proton resonance frequency shift (PRFS) to monitor high intensity focused ultrasound (HIFU) thermal ablations.Approach.Mechanical properties have been shown to be related to tissue damage induced by thermal ablations. Monitoring elasticity in addition to temperature changes may help in ensuring the efficacy and the accuracy of HIFU therapies. For this purpose, an MR-SSI method has been developed where the ultrasonic transducer is used for both mechanical wave generation and thermal ablation. Transient quasi-planar shear waves are generated using the acoustic radiation force, and their propagation is monitored in motion-sensitized phase MR images. Using a single-shot gradient-echo echo-planar-imaging sequence, MR images can be acquired at a sufficiently high temporal resolution to provide an update of PRFS thermometry and MR-SSI elastography maps in real time.Main results.The proposed method was first validated on a calibrated elasticity phantom, in which both the possibility to detect inclusions with different stiffness and repeatability were demonstrated. The standard deviation between the 8 performed measurements was 2% on the background of the phantom and 11%, at most, on the inclusions. A second experiment consisted in performing a HIFU heating in a gelatin phantom. The temperature increase was estimated to be 9 °C and the shear modulus was found to decrease from 2.9 to 1.8 kPa, reflecting the gel softening around the HIFU focus, whereas it remained steady in non-heated areas.Significance.The proposed MR-SSI technique allows monitoring HIFU ablations using thermometry and elastography simultaneously, without the need for an additional external mechanical exciter such as those used in MR elastography.

A new versatile MR-guided high-intensity focused ultrasound (HIFU) device for the treatment of musculoskeletal tumors.

Magnetic Resonance (MR) Imaging-guided High Intensity focused Ultrasound (MRgHIFU) is a non-invasive, non-ionizing thermal ablation therapy that is particularly interesting for the palliative or curative treatment of musculoskeletal tumors. We introduce a new modular MRgHIFU device that allows the ultrasound transducer to be positioned precisely and interactively over the body part to be treated. A flexible, MR-compatible supporting structure allows free positioning of the transducer under MRI/optical fusion imaging guidance. The same structure can be rigidified using pneumatic depression, holding the transducer rigidly in place. Targeting accuracy was first evaluated in vitro. The average targeting error of the complete process was found to be equal to 5.4 ± 2.2 mm in terms of focus position, and 4.7° ± 2° in terms of transducer orientation. First-in-man feasibility is demonstrated on a patient suffering from important, uncontrolled pain from a bone metastasis located in the forearm. The 81 × 47 × 34 mm3 lesion was successfully treated using five successive positions of the transducer, under real-time monitoring by MR Thermometry. Significant pain palliation was observed 3 days after the intervention. The system described and characterized in this study is a particularly interesting modular, low-cost MRgHIFU device for musculoskeletal tumor therapy.

An adaptive and fully automatic method for estimating the 3D position of bendable instruments using endoscopic images.

BACKGROUND: Flexible bendable instruments are key tools for performing surgical endoscopy. Being able to measure the 3D position of such instruments can be useful for various tasks, such as controlling automatically robotized instruments and analyzing motions. METHODS: An automatic method is proposed to infer the 3D pose of a single bending section instrument, using only the images provided by a monocular camera embedded at the tip of the endoscope. The proposed method relies on colored markers attached onto the bending section. The image of the instrument is segmented using a graph-based method and the corners of the markers are extracted by detecting the color transitions along Bézier curves fitted on edge points. These features are accurately located and then used to estimate the 3D pose of the instrument using an adaptive model that takes into account the mechanical play between the instrument and its housing channel. RESULTS: The feature extraction method provides good localization of marker corners with images of the in vivo environment despite sensor saturation due to strong lighting. The RMS error on estimation of the tip position of the instrument for laboratory experiments was 2.1, 1.96, and 3.18 mm in the x, y and z directions, respectively. Qualitative analysis in the case of in vivo images shows the ability to correctly estimate the 3D position of the instrument tip during real motions. CONCLUSIONS: The proposed method provides an automatic and accurate estimation of the 3D position of the tip of a bendable instrument in realistic conditions, where standard approaches fail.

A three-stage method for the 3D reconstruction of the tracheobronchial tree from CT scans.

This paper proposes a method for segmenting the airways from CT scans of the chest to obtain a 3D model that can be used in the virtual bronchoscopy for the exploration and the planning of paths to the lesions. The method is composed of 3 stages: a gross segmentation that reconstructs the main airway tree using adaptive region growing, a finer segmentation that identifies any potential airway region based on a 2D process that enhances bronchi walls using local information, and a final process to connect any isolated bronchus to the main airways using a morphologic reconstruction process and a path planning technique. The paper includes two examples for the evaluation and discussion of the proposal.

Control of postharvest diseases of fruit by heat and fungicides: efficacy, residue levels, and residue persistence. A review.

Extensive research has been done in recent years to reduce the heavy dependence on chemical fungicides to control postharvest diseases and disorders of horticultural crops. Alternative strategies were based on improved cultural practices, biological control, plant-defense promoters, and physical treatments such as UV illumination, radiofrequency treatment, heat therapy, and storage technologies. Among these, postharvest heat treatments such as hot water dips, short hot water rinsing and brushing, and hot air conditioning have reduced rot development and enhanced fruit resistance to chilling injury in sensitive cultivars while retaining fruit quality during cold storage and shelf life. Additive or synergistic increases in effectiveness were observed by integrating heat therapy with various chemical compounds, thus leading to significant reductions in the application of active ingredients to protect produce from decay. This paper highlights the knowledge on this topic with emphasis on heat therapy effects and factors affecting the uptake, persistence, and performance of fungicide residues when they are applied in combination with hot water.

Chlorpyrifos residues levels in fruits and vegetables after field treatment.

Chlorpyrifos (O,O-diethyl O-3,5,6-trichloro-2-pyridyl phosphorothioate) was applied with three different formulations on oranges, peaches, tomatoes, wine and table grapes, and its behaviour was evaluated after field treatment. The formulations applied were emulsifiable concentrates (EC), microencapsulates (ME), and wettable granules (WG). The residues were similar in all crops studied in the EC and WG experiments, except peaches with WG treatment, the residue amount was lower than EC values. Tomatoes which were grown in greenhouse showed similar residues in all treatments just after treatment. Wine and table grapes showed different decline curves in the EC experiments ascribable to the different growing technology. Instrumental limit of determination (LOD) and limit of quantification (LOQ) for all matrices were 0.01, and 0.03 mg kg⁻¹, respectively. Repeated treatments showed that Chlorpyrifos can accumulate leading to residue levels at the preharvest interval (PHI) over the maximum residue level (MRL), especially on oranges and peaches. Among the formulates used ME showed the higher risk of residues over the MRL at harvest.

Pesticides' influence on wine fermentation.

Wine quality strongly depends on the grape quality. To obtain high-quality wines, it is necessary to process healthy grapes at the correct ripeness stage and for this reason the farmer has to be especially careful in the prevention of parasite attacks on the grapevine. The most common fungal diseases affecting grape quality are downy and powdery mildew (Plasmopara viticola and Uncinula necator), and gray mold (Botrytis cinerea). On the other hand, the most dangerous insects are the grape moth (Lobesia botrana), vine mealybug (Planococcus ficus), and the citrus mealybug (Planococcus citri). Farmers fight grape diseases and insects applying pesticides that can be found at harvest time on grapes. The persistence of pesticides depends on the chemical characteristic of the active ingredients as well as on photodegradation, thermodegradation, codistillation, and enzymatic degradation. The pesticide residues on grapes can be transferred to the must and this can influence the selection and development of yeast strains. Moreover, yeasts can also influence the levels of the pesticides in the wine by reducing or adsorbing them on lees. During the fermentative process, yeasts can cause the disappearance of pesticide residues by degradation or absorption at the end of the fermentation when yeasts are deposited as lees. In this chapter, we reviewed the effect of commonly used herbicides, insecticides, and fungicides on yeasts. We also studied the effect of alcoholic and malolactic fermentation on pesticide residues.

Minor crops for export: a case study of boscalid, pyraclostrobin, lufenuron and lambda-cyhalothrin residue levels on green beans and spring onions in Egypt.

Dissipation rates of boscalid [2-chloro-N-(4' -chlorobiphenyl-2-yl)nicotinamide], pyraclostrobin [methyl 2-[1-(4-chlorophenyl) pyrazol-3-yloxymethyl]-N-methoxycarbanilate], lufenuron [(RS)-1-[2,5-dichloro-4-(1,1,2,3,3,3-hexafluoropropoxy)phenyl]-3-(2,6-difluorobenzoyl)urea] and lambda-cyhalothrin [(R)-cyano(3-phenoxyphenyl)methyl (1S,3S)-rel-3-[(1Z)-2-chloro-3,3,3-trifluoro-1-propenyl]-2,2-dimethylcyclopropanecarboxylate] in green beans and spring onions under Egyptian field conditions were studied. Field trials were carried out in 2008 in a Blue Nile farm, located at 70 kilometer (km) from Cairo (Egypt). The pesticides were sprayed at the recommended rate and samples were collected at pre-determined intervals. After treatment (T(0)) the pesticide residues in green beans were 7 times lower than in spring onions. This is due to a different structure of vegetable plant in the two crops. In spring onions, half-life (t(1/2)) of pyraclostrobin and lufenuron was 3.1 days and 9.8 days respectively. At day 14th (T(14)) after treatment boscalid residues were below the Maximum Residue Limit (MRL) (0.34 versus 0.5 mg/kg), pyraclostrobin and lambda -cyhalothrin residues were not detectable (ND), while lufenuron residues were above the MRL (0.06 versus 0.02 mg/kg). In green beans, at T(0), levels of boscalid, lufenuron and lambda -cyhalothrin were below the MRL (0.28 versus 2 mg/kg; ND versus 0.02 mg/kg; 0.06 versus 0.2 mg/kg, respectively) while, after 7 days treatment (T(7)) pyraclostrobin residues were above the MRL (0.03 versus 0.02 mg/kg). However, after 14 days the residue level could go below the MRL (0.02 mg/kg), as observed in spring onions.

Liquid chromatography electrospray ionization tandem mass spectrometric determination of quassin and neoquassin in fruits and vegetables.

Quassia amara wood chips are used by organic farmers as a valid alternative to synthetic insecticides. The powder of Q. amara contains high levels of quassin, neoquassin and picrasinoside B. In this study we developed a liquid chromatography mass spectrometry method for the rapid and accurate quantification of the insecticide quassinoids on fruits and vegetables. Quassinoids were extracted from fruits and vegetables with acetonitrile and separated on a Zorbax Column Eclipse XDB C8 by isocratic elution with a mobile phase consisting of water and methanol with 0.1% of formic acid. Using a high-performance liquid chromatograph coupled with an electrospray ionization tandem mass spectrometer (HPLC/ESI-MS/MS), quassinoids were selectively and simultaneously detected monitoring the multiple reaction (MRM) transitions of proton adduct precursor ions: m/z 389.5 --> 222.9 for quassin, 391.5 --> 372.9 for neoquassin and 576.1 --> 394.5 for picrasinoside B. For all quassinoids calibration was linear over a working range of 1 and 100 microg/kg with r > 0.991. Limit of determination (LOD) and limit of quantification (LOQ) for both quassinoids were 0.5 and 1 microg/kg respectively while for picrasinoside B they were 5 and 10 microg/kg. Quassinoid recoveries ranged from 85.3% to 105.3% with coefficients of variation between 2.5% and 12.8% for fruit and vegetables. The presence of interfering compounds in the fruit and vegetable extract was evaluated and found to be minimal. Due to the linear behavior it was concluded that the multiple reaction transitions of precursor ions can be used for analytical purposes, i.e. for identification and quantification of quassin, neoquassin, and picrasinoside B in fruit and vegetable extracts at trace levels.

Chemical variability, antifungal and antioxidant activity of Eucalyptus camaldulensis essential oil from Sardinia.

Essential oil (EO) from aerial parts of Eucalyptus camaldulensis Dehnh., growing wild in different localities of Sardinia (Italy), was extracted by steam distillation and analyzed by gas chromatography (GC) FID and GC-ion trap mass spectrometry (ITMS). The yields of EO (v/dry wt) ranged between 0.2-0.5%. Samples were harvested between April and December to study the seasonal chemical variability of the EO. The chemical composition varied depending on the different origins and showed strong fluctuation during the vegetative stage. Thirty-seven compounds, accounting for at least 97.7% of the total EOs were identified, the major components being p-cymene (27.8-42.7%), 1,8-cineole (4.1-39.5%), beta-phellandrene (3.9-23.8%), spathulenol (2.1-15.5%) and cryptone (3.2-10.2%). The oils possessed moderate amounts (1.4-4.7%) of two uncommon aldehydes, cuminal and phellandral. The essential oils were screened for their antifungal activities against common phytopathogenic fungi using the paper disk diffusion method and they showed activity at low doses against the fungi tested. The antioxidant activity, assessed by DPPH-test and expressed as Trolox equivalent antioxidant capacity, showed values ranging between 0.5 and 5.8 mmol/L.

Postinfection activity, residue levels, and persistence of azoxystrobin, fludioxonil, and pyrimethanil applied alone or in combination with heat and imazalil for green mold control on inoculated oranges.

The postinfection activity of azoxystrobin (AZX), fludioxonil (FLU), and pyrimethanil (PYR), applied alone or in combination with imazalil (IMZ), in controlling postharvest green mold in 'Salustiana' oranges inoculated with Penicillium digitatum was studied. Fruits were immersed for 30 or 60 s in (i) water or water mixtures at 20 degrees C containing AZX, FLU, or PYR at 600 mg/L; and (ii) IMZ at 600 mg/L, alone or in combination with AZX, FLU, or PYR at 600 mg/L. Similar treatments were performed at 50 degrees C using the active ingredients at half rates with respect to the treatments at 20 degrees C. Fungicide residues in fruits were analyzed following treatments and after 14 days of simulated shelf life at 17 degrees C. AZX or FLU mixtures at 20 degrees C for 30-60 s similarly but moderately reduced green mold decay with respect to control fruit; differences due to dip time were not significant. Superior control of decay was achieved by PYR and, especially, IMZ, applied alone or in combination with AZX, FLU, or PYR. The activity of PYR at 20 degrees C was significantly dependent on treatment time, whereas that of IMZ and combined treatments at 20 degrees C was not. The effectiveness of FLU or PYR mixtures at 50 degrees C in controlling decay was similar and superior to that of AZX. The action of single- or double-fungicide application was not dependent on dip time in most samples. IMZ or combined mixtures at 50 degrees C were consistently more effective with respect to single-fungicide treatments with AZX, FLU, or PYR. The application of heated fungicide mixtures resulted in significantly higher residue accumulation in most fruit samples compared to treatments performed at 20 degrees C. The degradation rate of fungicides was generally low and dependent on treatment conditions such as time, temperature, and the presence or not of other fungicides.

Floral markers of strawberry tree (Arbutus unedo L.) honey.

Strawberry tree honey, due to its characteristic bitter taste, is one of the most typical Mediterranean honeys, with Sardinia being one of the largest producers. According to specific chemical studies, homogentisic acid was identified as a possible marker of this honey. This work, based on HPLC-DAD-MS/MS analysis of strawberry tree (Arbutus unedo L.) honeys, previously selected by sensory evaluation and melissopalynological analysis, showed that, in addition to the above-mentioned acid, there were other high levels of substances useful for the botanical classification of this unifloral honey. Two of these compounds were isolated and identified as (+/-)-2-cis,4-trans-abscisic acid (c,t-ABA) and (+/-)-2-trans,4-trans-abscisic acid (t,t-ABA). A third compound, a new natural product named unedone, was characterized as an epoxidic derivative of the above-mentioned acids. Structures of c,t-ABA, t,t-ABA, and unedone were elucidated on the basis of extensive 1D and 2D NMR experiments, as well as HPLC-MS/MS and Q-TOF analysis. In selected honeys the average amounts of c,t-ABA, t,t-ABA, and unedone were 176.2+/-25.4, 162.3+/-21.1, and 32.9+/-7.1 mg/kg, respectively. Analysis of the A. unedo nectar confirmed the floral origin of these compounds found in the honey. Abscisic acids were found in other unifloral honeys but not in such high amount and with a constant ratio of about 1:1. For this reason, besides homogentisic acid, these compounds could be used as complementary markers of strawberry tree honey.

Influence of post-harvest application rates of cyprodinil, treatment time and temperature on residue levels and efficacy in controlling green mould on 'Valencia' oranges.

The effectiveness of heat treatments with water and cyprodinil in controlling post-harvest green mould caused by Penicillium digitatum was investigated on artificially inoculated 'Valencia' oranges. Residue levels of cyprodinil were determined in the oranges as a function of active ingredient concentration, temperature and treatment time. Cyprodinil residues were significantly dependent on treatment time when applied at 600 mg l(-1) and 20 degrees C, but not when fruit were treated at 150-300 mg l(-1). The application of cyprodinil at 50 or 100 mg l(-1) at 55 degrees C for 30 s produced similar residue levels, while residues increased when the application rate was 150 mg l(-1). Cyprodinil at 100 mg l(-1) and 60 degrees C produced a significant increase in residues compared to treatment at 50 mg l(-1); no significant increase in residues was found when the application rate was raised from 100 to 150 mg l(-1). In comparison to treatments performed at 20 degrees C, the application of a heated cyprodinil mixture resulted in significantly higher residues in fruit. All treatments with cyprodinil at 20 degrees C similarly reduced green mould after 7 days of storage at 20 degrees C. After 18 days, treatment with cyprodinil at 600 mg l(-1) for 30 s was more effective than at 150-300 mg l(-1). When dip time was extended to 90 or 180 s, treatment efficacy was positively related to fungicide concentration. Treatments with water at 55 degrees C for 30 s were as effective as cyprodinil at 50-100 mg l(-1), but less effective than cyprodinil at 150 mg l(-1). After 7 days, treatment with water or cyprodinil at 50-150 mg l(-1) and 60 degrees C were equally effective in controlling green mould; while, after 18 days, treatment with cyprodinil at 150 mg l(-1) was consistently more effective than at 50-100 mg l(-1) or hot water alone.

Flavonoid characterization and antioxidant activity of hydroalcoholic extracts from Achillea ligustica All.

Ethanolic extracts of Achillea ligustica All. (Asteraceae) flowering tops were evaluated. High-performance liquid chromatography-electrospray ionisation-mass spectrometry was used for the identification and quantification of phenolic compounds. 6-Hydroxykaempferol-3,6,4'-trimethyl ether, apigenin-6-C-glucoside-8-C-arabinoside, luteolin, and apigenin were the most abundant flavonoids. For the first time C-glycosylflavones were detected in A. ligustica with apigenin-6-C-glucoside-8-C-arabinoside being the most representative. The radical scavenging activity of the extracts was determined by DPPH test and ranged between 4.18 and 12.3 mM. The ability of these extracts to inhibit non-enzymatic lipid peroxidation was studied using the simple in vitro system of linoleic acid oxidation: five of the nine extracts exerted a protective effect at the lower amount tested (5 microg). Protection on CaCo-2 intestinal cells against TBH-induced toxicity was also investigated: the results showed that two of the extracts tested in this cell system had the ability to protect against oxidative stress induced by TBH starting from concentrations as low as 10 microg/ml.

Residue-free wines: fate of some quinone outside inhibitor (QoI) fungicides in the winemaking process.

The fate of three fungicide residues (fenamidone, pyraclostrobin, and trifloxystrobin) from vine to wine was studied to evaluate the decay ratio and the influence of the technological process. The aim of this work was to identify pesticides that can degrade rapidly or be eliminated together with byproduct (lees and cake) of the winemaking process to obtain wine free of residues. The disappearance rate on grapes was calculated as pseudo-first-order kinetics, and the half-life (t(1/2)) was in the range from 5.4 +/- 1.9 to 12.2 +/- 1.2 days. The mechanism of dissipation of the three quinone outside inhibitor (QoI) fungicides was studied using different model systems. It was observed that the main mechanism responsible for disappearance was photodegradation. For active ingredients (ai) the half-lives of fenamidone, pyraclostrobin, and trifloxystrobin were 10.2 +/- 0.8, 20.1 +/- 0.1, and 8.6 +/- 1.0 h, respectively, whereas for formulation higher half-lives were observed when epicuticular waxes were present (from 13.8 +/- 0.2 to 26.6 +/- 0.1 h). After winemaking, fenamidone, pyraclostrobin, and trifloxystrobin residues were not detected in the wine, but they were present in the cake and lees. This was due to the adsorption of pesticide residues to the solid parts, which are always eliminated at the end of the alcoholic fermentation. The data obtained in these experiments suggest that these three active ingredients could be used in a planning process to obtain residue-free wines.

Persistence of two neem formulations on peach leaves and fruit: effect of the distribution.

Persistence of azadirachtins (A+B) and of the other limonoids (nimbin, salannin, deacetylnimbin, and deacetylsalannin) on peach leaves and fruits was studied using a commercial formulation (form. C) compared with an experimental formulation (form. E) prepared with coformulations allowed in organic culture. Field experiments were carried out using three concentrations: 1x, 5x, and 10x the dose recommended by the manufacturer. The EU maximum residue level (MRL) in fruits and vegetables for azadirachtin A is 1 mg/kg with a preharvest interval (PHI) of 3 days. At the recommended dose, azadirachtin A residue on fruits was not detectable (LOQ < 0.8 microg/kg). After field treatment at the 5x concentration, azadirachtoids were found with 22% in the epicuticular waxes and the remaining 78% on the fruit surface. No residues were found in the fruit pulp. The experimental formulation (E) produced lower residues on leaves and fruit compared with the commercial formulation (C), although formulation E showed greater stability. This is probably due to the amount of the active ingredients that diffuse into the epicuticular wax layer thus enhancing photostability of azadirachtoids.

Methyl syringate: a chemical marker of asphodel (Asphodelus microcarpus Salzm. et Viv.) monofloral honey.

During the liquid chromatographic study of the phenolic fraction of monofloral honeys was detected in the asphodel honey ( Asphodelus microcarpus Salzm. et Viv.) chromatogram a distinctive peak not detected in other monofloral honeys such as Arbutus unedo L., Hedysarum coronarium , Eucalyptus spp., and Galactites tomentosa . After thin layer chromatography (TLC) purification and characterization by NMR and LC-MS/MS, the compound was identified as methyl syringate (MSYR) and confirmed against an original standard. Levels of MSYR were measured in honeys of 2005, 2006, and 2007 by HPLC-DAD analysis. Level determination of MSYR was repeated in 2008 for 2006 and 2007 honeys to evaluate chemical stability of this phenolic compound. Levels of MSYR measured 1 year after the sampling did not show significant statistical differences (p < 0.05). The stability of MSYR was also confirmed by 12 asphodel honey samples collected in 2005 that showed amounts of methyl syringate comparable with those found in fresh honey. For the evaluation of MSYR origin, samples of nectars were collected from flowers and the content of MSYR was measured. Levels of MSYR in honeys are originated from the nectar with an average contribution of the nectar to the honey of 80%. Melissopalinological analysis did not allow the attribution of the honey monofloral origin because levels of asphodel pollen were <6% for all analyzed samples. Previously reported levels of MSYR for robinia, rape, chestnut, clover, linden blossom, dandelion, sunflower, thyme, manuka, and fir honeys were <5 mg/kg. For this reason, a minimum level of 122.6 mg/kg for MSYR in asphodel honeys can be considered as a chemical marker and, unlike the melissopalynological analysis, can be used for the origin attribution and to evaluate the percent of asphodel nectar in the honey.

Fate of azadirachtin A and related azadirachtoids on tomatoes after greenhouse treatment.

The degradation of the main azadirachtoids on tomatoes was studied after greenhouse treatment. These experiments were carried out at 1 and 5x the concentration recommended by the manufacturer. In all experiments the deposition of azadirachtin A (AZA-A) was below the maximum residue level (MRL). Even if at the highest dose, AZA-A half-life time calculated as pseudo first order kinetic was 1.2 days in agreement with the recommended preharvest interval (PHI) of 3 days. Experiments with a model system showed that sunlight photodegradation is the main factor influencing the rate of disappearance of AZA-A after greenhouse treatment while tomato epicuticular waxes doubled the photodegradation rate of AZA-A in a commercial formulation.