Development of a Qualitative Test to Detect the Presence of Organophosphate Pesticides on Fruits and Vegetables.

BACKGROUND: In recent decades, the use of pesticides in agriculture has increased at a fast pace, highlighting safety problems for the environment and human health, which in turn has made it necessary to develop new detection and decontamination systems for pesticides. METHODS: A new qualitative test capable of detecting the presence of pesticides on fruits and vegetables by using thermostable enzymes was discovered, and the test was carried out on apples and aubergines. The contaminating pesticides were extracted from fruits with acetonitrile and analyzed with a biosensor system based on the thermostable esterase EST2 immobilized on a nitrocellulose filter. This enzyme is irreversibly inhibited mainly in the presence of organophosphates pesticides. Therefore, by observing esterase activity inhibition, we revealed the presence of residual pesticides on the fruits and vegetables. RESULTS: By analyzing the rate of esterase activity inhibition, we predicted that residual pesticides are present on the surface of the fruits. When we cleaned the fruits by washing them in the presence of the phosphotriesterase SsoPox before the detection of the esterase activity on filters, we observed a full recovery of the activity for apples and 30% for aubergines, indicating that the enzymatic decontamination of organophosphates pesticides took place. CONCLUSIONS: The reported method permitted us to assess the pesticides present on the vegetables and their decontamination.

Endogenous and Exogenous Regulatory Signaling in the Secretory Pathway: Role of Golgi Signaling Molecules in Cancer.

The biosynthetic transport route that constitutes the secretory pathway plays a fundamental role in the cell, providing to the synthesis and transport of around one third of human proteins and most lipids. Signaling molecules within autoregulatory circuits on the intracellular membranes of the secretory pathway regulate these processes, especially at the level of the Golgi complex. Indeed, cancer cells can hijack several of these signaling molecules, and therefore also the underlying regulated processes, to bolster their growth or gain more aggressive phenotypes. Here, we review the most important autoregulatory circuits acting on the Golgi, emphasizing the role of specific signaling molecules in cancer. In fact, we propose to draw awareness to highlight the Golgi-localized regulatory systems as potential targets in cancer therapy.

Enzyme Promiscuous Activity: How to Define it and its Evolutionary Aspects.

Enzymes are among the most studied biological molecules because better understanding enzymes structure and activity will shed more light on their biological processes and regulation; from a biotechnological point of view there are many examples of enzymes used with the aim to obtain new products and/or to make industrial processes less invasive towards the environment. Enzymes are known for their high specificity in the recognition of a substrate but considering the particular features of an increasing number of enzymes this is not completely true, in fact, many enzymes are active on different substrates: this ability is called enzyme promiscuity. Usually, promiscuous activities have significantly lower kinetic parameters than to that of primary activity, but they have a crucial role in gene evolution. It is accepted that gene duplication followed by sequence divergence is considered a key evolutionary mechanism to generate new enzyme functions. In this way, promiscuous activities are the starting point to increase a secondary activity in the main activity and then get a new enzyme. The primary activity can be lost or reduced to a promiscuous activity. In this review we describe the differences between substrate and enzyme promiscuity, and its rule in gene evolution. From a practical point of view the knowledge of promiscuity can facilitate the in vitro progress of proteins engineering, both for biomedical and industrial applications. In particular, we report cases regarding esterases, phosphotriesterases and cytochrome P450.

Comparison of corneal densitometry between big-bubble and visco-bubble deep anterior lamellar keratoplasty.

AIMS: To evaluate deep corneal densitometry and visual outcomes after big-bubble (BB-DALK) and visco-bubble (VB-DALK) deep anterior lamellar keratoplasty performed in patients with keratoconus. METHODS: Prospective comparative study of 50 advanced keratoconic patients who underwent DALK surgery; 25 eyes (group I) were completed with BB-DALK and 25 eyes (group II) with VB-DALK after the failure of pneumatic dissection. Best spectacle-corrected visual acuity (BSCVA), corneal tomographic parameters and endothelial cell count were recorded 1, 3, 6, 12 and 24 months after surgery. Densitometric analysis of the deep corneal interface was obtained using Scheimpflug tomography at each visit; values recorded were compared between the two groups and statistically analysed. RESULTS: BSCVA was significantly better in the BB-DALK group than the VB-DALK group (0.39±0.29 vs 0.65±0.23 logarithm of the minimum angle of resolution, respectively) for the first 3 months; and in the same time period, densitometry was significantly higher in the VB-DALK group than those recorded in the BB-DALK group (23.97±5.34 vs 17.13±4.44 grayscale units). However, densitometric values and visual acuity did not differ significantly in the two groups at 1 year. No statistically significant difference for the other variables analysed at any time frame was found. CONCLUSION: The use of viscoelastic substance in the VB-DALK technique may induce modification of interface stromal reflectivity resulting in reduced visual acuity up to 3 months postoperatively. However, this initial negative effect on the interface quality does not affect the long-term visual outcome, with densitometric values and visual outcomes similar in the two groups from 6 months postoperatively.

Efficacy and Safety of Wet Wipes Containing Hy-Ter® Solution Compared with Standard Care for Bilateral Posterior Blepharitis: A Preliminary Randomized Controlled Study.

INTRODUCTION: A novel treatment for posterior blepharitis which involves the use of disposable wet wipes imbibed with a solution of terpinen-4-ol and hyaluronic acid (Hy-Ter®) as well as aloe, natural anti-inflammatories and antiseptics (Blephapad Combo; Novelty Technology Care, Milan, Italy) has been proposed. The aim of this study was to compare the efficacy and safety of treatment with Blephapad Combo with standard treatment. METHODS: We conducted 4-week, open-label, randomized controlled study with a closed sequential design at the Magna Graecia University of Catanzaro, Italy in which patients aged > 40 years with symmetrical bilateral posterior blepharitis were enrolled. Each eye represented a single experimental unit. Patients were randomly assigned using a computer-generated randomization list to apply a reusable heated compress to the eyelid of one eye before cleansing the eyelid with the Blephapad Combo wet wipe (Blephapad Combo treatment arm) or to use the standard treatment of applying a wet and warm gauze to the other eye (standard treatment arm), twice daily for 4 weeks. The primary endpoint was the percentage change from baseline to week 4 in meibomian gland dysfunction (MGD) grading scale scores. RESULTS: Eighteen patients (9 men; 9 women) with a mean (± standard deviation) age of 66.9 ± 9.03 years were included in the study. Compared with the eye receiving the standard treatment, treatment with Blephapad Combo appeared to improve the MGD total score after 4 weeks of treatment (mean change from baseline - 29.9 vs. - 38.5%). The assessment of the investigators was that in 11 patients the eye treated with Blephapad Combo showed greater benefit, in two patients the eye treated with the standard treatment showed greater benefit and in four patients there was no difference between treatments. Blephapad Combo was well tolerated, with no serious adverse events (AEs) reported. Minor ocular AEs were reported in 44.4 and 38.9% of patients in the Blephard Combo and standard treatment arms, respectively. CONCLUSIONS: Treatment with Blephapad Combo was more effective than the standard treatment in ameliorating MGD in patients with posterior blepharitis. Minor ocular AEs events were equally distributed between the two treatments arms. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT03301844.

Innovative Biocatalysts as Tools to Detect and Inactivate Nerve Agents.

Pesticides and warfare nerve agents are frequently organophosphates (OPs) or related compounds. Their acute toxicity highlighted more than ever the need to explore applicable strategies for the sensing, decontamination and/or detoxification of these compounds. Herein, we report the use of two different thermostable enzyme families capable to detect and inactivate OPs. In particular, mutants of carboxylesterase-2 from Alicyclobacillus acidocaldarius and of phosphotriesterase-like lactonases from Sulfolobus solfataricus and Sulfolobus acidocaldarius, have been selected and assembled in an optimized format for the development of an electrochemical biosensor and a decontamination formulation, respectively. The features of the developed tools have been tested in an ad-hoc fabricated chamber, to mimic an alarming situation of exposure to a nerve agent. Choosing ethyl-paraoxon as nerve agent simulant, a limit of detection (LOD) of 0.4 nM, after 5 s of exposure time was obtained. Furthermore, an optimized enzymatic formulation was used for a fast and efficient environmental detoxification (>99%) of the nebulized nerve agent simulants in the air and on surfaces. Crucial, large-scale experiments have been possible thanks to production of grams amounts of pure (>90%) enzymes.

Results of viscobubble deep anterior lamellar keratoplasty after failure of pneumatic dissection.

AIM: To report the outcomes of bubble formation obtained by means of intrastromal injection of ophthalmic viscoelastic device (OVD) after failure of pneumatic dissection for deep anterior lamellar keratoplasty (DALK). METHODS: DALK was performed in 140 keratoconic eyes of 130 patients by injecting air and OVD only secondarily, after pneumatic dissection had failed; the bubble formation rates after air and OVD injection were recorded; complications, best spectacle-corrected visual acuity (BSCVA) and corneal tomographic parameters were evaluated 3 months, 6 months and 12 months postoperatively, as well as after complete suture removal. RESULTS: Air injection created a big bubble in 106/140 eyes (75.71%); OVD injection was not attempted in 4 eyes (perforation during cannula insertion n=2; air bubble burst n=2) and created a big bubble in 28 of the remaining 30 eyes (93.33%, 20% of the total). Manual dissection was required in 2/30 eyes (6.66%, 1.42% of the total) after failed OVD-assisted dissection.Deep folds, interface opacity and reduced BSCVA were noted in both eyes after failed OVD-assisted dissection. BSCVA was statistically better after pneumatic-assisted than after OVD-assisted dissection (P 0.01) only up to 3 months postoperatively; no statistically significant differences were recorded between the two techniques at later examinations. CONCLUSION: Intrastromal injection of OVD after failed pneumatic dissection increases considerably the success rate of bubble formation (from 75.71% to 95.71% in our series); however, when bubble formation fails, infiltration of OVD into the residual stroma makes manual dissection particularly challenging and causes severe interface haze resulting in poor visual outcomes.

Results of Descemet Stripping Automated Endothelial Keratoplasty for the Treatment of Late Corneal Decompensation Secondary to Obstetrical Forceps Trauma.

PURPOSE: To describe the clinical outcomes of Descemet stripping automated endothelial keratoplasty (DSAEK) in a series of patients affected by progressive endothelial cell loss leading to corneal decompensation with Descemet membrane (DM) breaks caused by obstetrical forceps trauma. METHODS: Seven amblyopic eyes of 7 patients affected by unilateral visual loss due to increasing corneal edema were included in the study. In all eyes, slit-lamp examination revealed typical DM breaks and diffuse corneal edema: these findings were compatible with a history of obstetrical forceps-assisted delivery. DSAEK was performed in all cases (in 3 eyes combined with phacoemulsification and intraocular lens implantation); visual acuity, refraction, corneal topography, and endothelial cell density were recorded preoperatively and 1 year after surgery. RESULTS: Visual acuity improved in all cases by at least 2 Snellen lines; topographic astigmatism lower than 2.5 diopters was associated with better visual recovery. No intraoperative or postoperative complications were reported, and the corneas remained clear throughout the follow-up. CONCLUSIONS: In eyes with endothelial decompensation secondary to DM breaks caused at birth by forceps injury, DSAEK can be performed uneventfully and restore cornea clarity. However, visual recovery is limited by the preexistent amblyopia, usually resulting from anisometropia and/or high-degree unilateral astigmatism. The results obtained in our patients suggest that low-degree astigmatism may be associated with better visual outcomes.

Single and repeated baclofen treatment attenuates the discriminative stimulus effects of morphine in rats.

The GABA(B) agonists block the rewarding properties of opiates. However, the role of GABA(B) receptors in the discriminative properties of these drugs has received little attention. In this line, the present study was performed to investigate the effects of acute (Experiment 1) and chronic (Experiment 2) pretreatment with baclofen on the discriminative stimulus effects of morphine. Rats were trained to discriminate morphine (5mg/kgi.p.) from saline under a two-lever fixed-ratio schedule of food reinforcement. Experiment 1: a morphine generalization curve was obtained under standard conditions in control and baclofen (1 and 2mg/kgi.p.) pretreated animals. Acute baclofen pretreatment attenuated morphine-lever responding and response rate at both doses. Experiment 2: the animals were randomly divided in two groups and a morphine generalization curve was obtained in daily consecutive test sessions before (TEST1) and after (TEST2) chronic saline (Group I) or chronic baclofen (2mg/kg) (Group II) administration. As expected, chronic saline pretreatment was ineffective, while chronic baclofen pretreatment attenuated the stimulus properties of morphine, without modifying the rate-decreasing effect of the drug.The data support a role for GABAergic neurotransmission in the discriminative effects of opiates and demonstrate that a short-term treatment with baclofen is useful for decreasing the sensitivity to narcotic cue.

D-cycloserine inhibits the development and the expression of locomotor sensitization to amphetamine in rats.

Amphetamine use is now an epidemic of global proportions; however, no approved medications are available to treat amphetamine abusers. Since glutamate plays a crucial role in regulating the development and expression of addictive behaviors, such as sensitization, in the present study we examined the possible effect of D-cycloserine, a partial agonist at the glycine site of N-methyl-D-aspartate receptors, on the development and expression of locomotor sensitization to amphetamine. Rats were intraperitoneally injected with saline or D-cycloserine (6 mg/kg), before saline or amphetamine (1.5 mg/kg), on days 1-10. Three days after ceasing the chronic treatment, a challenge test was run. The animals received saline or 6 mg/kg D-cycloserine and, 1 h later, they received 0.75 or 1.5 mg/kg amphetamine; motility was recorded for 2 h. The chronic treatment with D-cycloserine alone had no influence on the acute behavioral response to amphetamine. However, D-cycloserine prevented the development and inhibited the expression of amphetamine sensitization; in both cases the D-cycloserine effect was not significantly different as a function of amphetamine challenge dose. As behavioral sensitization is proposed to model the enhanced 'drug wanting' observed in psychostimulant abusers, the present data suggest that D-cycloserine might be a useful therapeutic approach to its treatment.

Rapid prototyping: a new method of preparing trays for indirect bonding.

This article describes a new method of preparing trays for indirect bracket bonding. Computer-aided technology is used to design the individualized trays, which are then produced with a rapid prototyping procedure. Application in clinical practice and the advantages of time savings and accurate bracket placement are discussed.

A new one-step dental flowable composite for orthodontic use: an in vitro bond strength study.

A new flowable composite, Denfil Flow, has shown an acceptable shear bond strength for bonding orthodontic brackets, when used with an intermediate, unfilled, low-viscosity resin. According to the manufacturer, it also shows a good viscosity for use with no preliminary adhesive. This could reduce the total time of bonding procedure while maintaining clinically useful bond strength. The aim of the current research was to assess this property. Eighty extracted human premolars were randomly divided into four equal groups. Stainless steel brackets were bonded to etched enamel using (1) Denfil Flow, (2) a traditional flowable composite (Dyract Flow), (3) Denfil Flow composite resin and an intermediate liquid resin, and (4) Transbond XT adhesive. Debonding was performed with a shearing force. The residual adhesive on the enamel surface was evaluated using the adhesive remnant index. The bond strength of Denfil Flow (34.8 MPa) showed no significant difference with the other control groups and was clinically acceptable. Denfil Flow and Dyract Flow tended to display cohesive failure within the adhesive. Denfil Flow can be used without liquid resin to reduce the bonding procedure time while maintaining acceptable bond strength. Further studies are required to evaluate the enamel surface of the teeth after the same polishing procedure in the four groups.