Development of a haemolytic-enzymatic assay with mediated amperometric detection for palytoxin analysis: application to mussels.

An electrochemical sensor for palytoxin (PlTX) detection, based on a strip of eight screen-printed electrodes connected to a cost-effective and portable apparatus, is reported. Sheep erythrocytes were used to test the palytoxin detector and degree of haemolysis was evaluated by measuring release of the cytosolic lactate dehydrogenase (LDH). Percentage haemolysis and, therefore, the amount of LDH measured, by use of NADH/pyruvate and appropriate electrochemical mediators, was correlated with the concentration of the toxin. Two different electrochemical approaches were investigated for evaluation of LDH release, but only one based on the use of a binary redox mediator sequence (phenazine methosulfate in conjugation with hexacyanoferrate(III)) proved useful for our purpose. After analytical and biochemical characterization, the sensor strip was used to measure palytoxin. Sheep blood and standard solutions of PlTX were left to react for two different incubation times (24 h or 4 h), resulting in working ranges of 7 × 10(-3)-0.02 ng mL(-1) and 0.16-1.3 ng mL(-1), respectively. The specificity of the test for palytoxin was evaluated by use of ouabain, which acts in the same way as PlTX on the Na(+)/K(+)-ATPase pump. A cross-reactivity study, using high concentrations of other marine biotoxins was also conducted. Experiments to evaluate the matrix effect and recovery from mussels are discussed.

Superficial radiotherapy for multiple keratoacanthomas.

We report the case of a 76-year-old Caucasian woman who attended our clinic with multiple keratoacanthomas. Radiotherapy was considered a viable and effective option in such an old patient, who could not be a good surgical candidate for number and distribution of the lesions, and for the age. After a 12-month follow-up, we observed the complete and global disappearance of the lesions; the patient was therefore very satisfied especially in view of the clinical outcome. According to our point of view, radiotherapy allows the physician to obtain a good oncological radicality and excellent cosmetic results too.

Severe obstructive sleep apnoea syndrome and erectile dysfunction: a prospective randomised study to compare sildenafil vs. nasal continuous positive airway pressure.

BACKGROUND: A high incidence of erectile dysfunction (ED) among patients with obstructive sleep apnoea syndrome (OSAS) has been reported, with a strong correlation between obstructive sleep apnoea, ED, and quality of life (QOL), and it has been estimated that 10-60% of patients with OSAS suffer from ED. In this prospective randomised controlled trial, we investigated 82 men with ED consecutively who were referred to the outpatient clinic for sleep disorders and had severe OSAS (AHI> 30 events/h) without any other comorbidities as a possible cause of ED. The aim of this study was to evaluate and compare the efficacy of sildenafil vs. continuous positive airway pressure (CPAP) in men with ED and severe OSAS. METHODS: Eighty-two patients were randomised to two main treatment groups: group 1 patients (n = 41) were treated with 100-mg sildenafil 1 h before sexual intercourse without CPAP, and group 2 patients (n = 41 men) were treated with only nasal CPAP during night time sleep. Both groups were evaluated with the same questionnaires (International Index of Erectile Function-EF domain; Sex Encounter Profile; Erectile Dysfunction Inventory Treatment Satisfaction) 12 weeks after treatment. RESULTS: In patients receiving sildenafil treatment, 58.2% of those who attempted sexual intercourses were successful compared to 30.4% in the CPAP group. The mean number of successful attempts per week was significantly higher in the sildenafil group compared with the CPAP group (2.9 vs. 1.7, respectively; p < 0.0001). The mean IIEF-EF domain scores were significantly higher in the sildenafil group compared with the CPAP group (p < 0.0001). The overall satisfaction rate was 68% with sildenafil treatment and 29% with CPAP treatment. CONCLUSIONS: This study confirms that severe OSAS is strongly associated with erectile dysfunction. CPAP and sildenafil (100 mg) are safe and effective therapies for OSAS-related ED patients. In the present study sildenafil was more effective than CPAP in treating ED associated with OSAS, as indicated by a significantly higher rate of successful attempts at intercourse and higher IIEF-EF domain scores. Our study, to date, is the only that has investigated sildenafil in patients with severe OSAS.

A prospective study to evaluate the efficacy of Cistiquer in improving lower urinary tract symptoms in females with urethral syndrome.

AIM: The aim of the study was to compare Cistiquer, a new phytotherapeutic product developed for chronic bladder inflammatory diseases, and intra-vesical administration of gentamicin plus betametasone, in females with urethral syndrome. METHODS: Between september 2013 and may 2014, 60 women with urethral syndrome and trigonitis were incuded in this study. Patients were randomly assigned to treatment with intra-vesical administration of betametasone 8 mg plus gentamicin 80 mg (group A), and oral administration of Cistiquer (group B) for 7 weeks. Before and after the therapeutic protocol, symptoms were assessed by three days voiding diary, the overactive bladder questionnaire short form and a ten points visual analogic scale adopted to assess the micturition discomfort. Histologic findings were assessed by the examination of specimens obtained by cold bladder biopsies of the bladder trigone at baseline in all the subjects. RESULTS: The two groups had significant and comparable symptoms improvement. However, the score obtained from the visual analogic scale decreased significantly only in the group submitted to oral therapy. Furthermore, in the group treated with endovesical approach, higher drop out rate and higher incidence of urinary infection were observed. CONCLUSION: Patients with urethral syndrome and trigonitis improved symptoms either with oral therapy with Cistiquer and with intra-vesical administration of gentamicin plus betametasone. However, treatment adherence resulted higher for patients treated by oral therapy and rate of adverse events resulted higher for those submitted to endovesical treatment.

Carbon nanostructured materials for applications in nano-medicine, cultural heritage, and electrochemical biosensors.

This review covers applications of pristine and functionalized single-wall carbon nanotubes (SWCNTs) in nano-medicine, cultural heritage, and biosensors. The physicochemical properties of these engineered nanoparticles are similar to those of ultrafine components of airborne pollution (UF) and might have similar adverse effects. UF may impair cardiovascular autonomic control (inducing a high-risk condition for adverse cardiovascular effects), cause mammalian embryo toxicity, and increase geno-cytotoxic risk. SWCNTs coated with a biopolymer, for example polyethylenimine (PEI), become extremely biocompatible, hence are useful for in-vivo and in-vitro drug delivery and gene transfection. It is also possible to successfully immobilize a human enteric virus on PEI/SWCNT composites, suggesting application as a carrier in non-permissive media. The effectiveness of carbon nanostructured materials in the cleaning, restoration, and consolidation of deteriorated historical surfaces has been widely shown by the use of carbon nanomicelles to remove black dendritic crust from stone surfaces. The nanomicelles, here, have the twofold role of delivery and controlled release of the cleaning agents. The high biocompatibility of functionalized SWCNTs with enzymes and proteins is a fundamental feature used in the assembly of electrochemical biosensors. In particular, a third-generation protoporphyrin IX-based biosensor has been assembled for amperometric detection of nitrite, an environmental pollutant involved in the biodeterioration and black encrustation of historical surfaces.

Graphene oxide nanoribbons (GNO), reduced graphene nanoribbons (GNR), and multi-layers of oxidized graphene functionalized with ionic liquids (GO-IL) for assembly of miniaturized electrochemical devices.

In this critical review, new nanomaterials based on graphene (GN) are described, especially those used for the assembly of miniaturized electrochemical transducers. In particular, the physicochemical properties and mechanical features of few layers of graphene (FLGs) are described, as is their use for assembly of chemically modified sensors, biosensors, and immunosensors. The FLGs described here were functionalized by chemical treatment in solution, resulting in oxidized and/or reduced surfaces, edges, and sides. The presence of oxygenated functionality strongly affects the electrocatalysis and the electron-transfer properties of several molecular targets, not only in the solid phase (e.g. in field-effect transistors, FETs) but also in liquid matrices (chemically modified electrodes and biosensors). In addition, "green chemistry" reagents, for example ionic liquids (ILs) can be used for exfoliation and intercalation of graphene planes, to obtain stable and homogeneous nanodispersions. The assembled sensors, biosensors, and immunosensors are extremely useful for electrochemical detection of several electro-active targets of importance in food analysis, environmental monitoring, and clinical diagnosis. A detailed description of each analytical application has been given in this critical review and brief remarks on the emerging disciplines of nanomedicine and nanofoods are also discussed.

Towards the development of a single-step immunosensor based on an electrochemical screen-printed electrode strip coupled with immunomagnetic beads.

This work investigates the behaviour of two alternative systems that model the crucial event involved in any ELISA test, i.e. the molecular recognition between an antigen and its specific antibody on a solid phase, and its measurement. Each approach is devised with the goal of making possible a single-step, separation and wash-free amperometric magneto-immunosensor. Magnetic particles (MBs) are used as support for the immobilization of rabbit IgGs that are recognized by the specific anti-rabbit IgG-HRP. The assay protocol is based on the use of a series of small "reservoirs" containing phosphate buffer, hydroquinone, anti-rabbit IgG-HRP and an appropriate amount of MB-rabbit IgG. After a brief incubation, the content of each "reservoir" is transferred to one of the wells of a 8-well magnetized-screen-printed electrode strip. The resulting MB-IgG-anti-IgG-HRP chain, is then concentrated on the working electrode surface for electrochemical measurement. Two different approaches to monitor this immunological reaction are investigated. The first one is based on the enzyme-channeling principle (ECP) and involves the use of a second enzyme, glucose oxidase (GOD), immobilized on the working electrode previously modified with Prussian Blue. Since the H(2)O(2) produced by GOD is the co-substrate of the HRP enzyme, glucose is added into the well and the current, generated by the residual H(2)O(2), is measured. The second, more direct, approach is performed without exploiting ECP (no GOD enzyme), by adding H(2)O(2) into the well and measuring the current generated by the HRP product on a pristine screen-printed electrode. Both approaches yielded a typical sigmoidal binding curve, illustrating the discrimination between the signal produced by the immuno-bound HRP concentrated on the electrode surface, and the background signal due to HRP in the bulk solution.

Oxidized graphene in ionic liquids for assembling chemically modified electrodes: a structural and electrochemical characterization study.

Dispersions of graphene oxide (GO) nanoribbons in ionic liquids, ILs (either 1-butyl-3-methylimidazolium chloride (BMIM-Cl-) or 1-butylpyridinium chloride (-Bupy-Cl-)) have been used to assemble modified screen printed electrodes (SPEs). The graphene oxide/ionic liquid dispersions have been morphologically and structurally characterized by the use of several techniques: X-ray photoelectron spectroscopy (XPS), Fourier transform-infrared (FT-IR) spectroscopy, high-resolution-transmission electron microscopy (HR-TEM). The assembled modified SPEs have then been challenged with various compounds and compared to several electro-active targets. In all cases high peak currents were detected, as well as significant potential shifts, especially in the detection of catecholamines and NADH, compared with the bare SPE and the conventional electrodes, such as glassy carbon (GC) and highly oriented pyrolitic graphite (HOPG). This opens the way to the assembly of new types of sensors and biosensors. The enhanced performances observed are attributed to electrocatalytic effects related to the high electrode surface area, to oxygen-assisted electron transfer, as well as to the disordering effect of the ILs, this latter related to the favorable π-π interactions with the ILs and the GO plane.

A prospective randomized study to compare pelvic floor rehabilitation and dapoxetine for treatment of lifelong premature ejaculation.

Premature ejaculation (PE) is the most common male sexual disorder. We compared pelvic floor muscle rehabilitation to on-demand treatment with the selective serotonin reuptake inhibitor dapoxetine in 40 men with lifelong PE (baseline intra-vaginal ejaculatory latency time (IELT) ≤1 min). Subjects were randomized into the following two treatment groups: (1) PFM rehabilitation or (2) 30 or 60 mg of on-demand dapoxetine. Total treatment time for both groups was 12 weeks, at the end of which, IELT mean values were calculated to compare the effectiveness of the two different therapeutic approaches. At the end of treatment, 11 of the 19 patients (57%) treated with rehabilitation were able to control the ejaculation reflex, with a mean IELT of 126.6 sec (range: 123.6-152.4 sec). In the dapoxetine group, after 12 weeks of therapy, 5 of 8 (62.5%) patients in the 30 mg subgroup and five of seven (72%) in the 60 mg subgroup had an IELT >180 sec (mean: 178.2 and 202.8 sec, respectively). The results obtained in the group treated with pelvic floor rehabilitation are promising, and this treatment represents an important cost reduction if compared to dapoxetine on-demand treatment. The present study confirms the data that are previously available in the literature on the efficacy and safety of the new inhibitor of serotonin reuptake, dapoxetine, as well as proposes and evaluates a new type of physical treatment that may be a viable therapeutic option for treatment of PE.

Respiratory depression after administration of single-dose neuraxial morphine for post-cesarean delivery analgesia: a retrospective cohort study.

BACKGROUND: Neuraxial administration of long-acting opioid is the "gold standard" for the management of postoperative pain following cesarean delivery. Respiratory depression, however, remains a concerning complication. METHODS: This retrospective single-center study of 4963 patients evaluated the frequency of respiratory depression after neuraxial morphine administration in a post-cesarean delivery population. The spinal dose of morphine varied from 100 to 450 µg intrathecally, and from 3 to 5 mg epidurally. The primary outcome was the initiation of a Rapid Response Team (RRT) event for respiratory failure due to neuraxial opioid in the 24 h following morphine administration. Secondary outcomes studied included oxygen desaturation events (SpO2 <90%), initiation of oxygen therapy and naloxone administration. RESULTS: There were no respiratory RRT events within the study period (95% confidence interval [CI] 0 to 7 per 10 000). There were no desaturation events recorded and no patients received supplemental oxygen therapy or naloxone (95% CI 0 to 7 per 10 000). CONCLUSION: Clinically significant respiratory depression is rare among patients receiving neuraxial morphine for post-cesarean delivery analgesia.

A bienzyme electrochemical probe for flow injection analysis of okadaic acid based on protein phosphatase-2A inhibition: an optimization study.

A bienzyme electrochemical probe has been assembled and used to monitor the inhibition of the enzyme protein phosphatase-2A (PP2A) by okadaic acid (OA), taking advantage of the particular characteristics of a biochemical pathway in which PP2A is involved. This enzyme has significant activity toward glycogen phosphorylase a (PHOS a), which in turn catalyzes the conversion of glycogen to glucose-1-phosphate (G-1-P). In addition, PP2A is strongly inhibited by OA and its derivatives. Due to this combination of properties, PP2A was employed to develop an assay system involving a preliminary phase of off-line enzymatic incubations (OA/PP2A, PP2A/PHOS a, PHOS a/glycogen+phosphate). This off-line step was followed by the electrochemical detection of H2O2, which is the final product of two sequential enzymatic reactions: G-1-P with alkaline phosphatase (AP) producing glucose, then glucose with glucose oxidase (GOD) producing hydrogen peroxide. These two enzymes were coimmobilized on a nylon net membrane that was placed over an H2O2 platinum probe inserted into a flow injection analysis (FIA) system. During a first phase of the study, all analytical parameters were optimized. During a subsequent phase, the inhibition of PP2A enzyme by OA was evaluated. The calibration of the system shows a working range for detection of OA between 30 and 250 pg ml(-1). The total analysis time is the sum of 50 min for the off-line enzymatic incubations and 4 min for the biosensor response.

Origami multiple paper-based electrochemical biosensors for pesticide detection.

Herein, we propose the first three-dimensional origami paper-based device for the detection of several classes of pesticides by combining different enzyme-inhibition biosensors. This device was developed by integrating two different office paper-based screen-printed electrodes and multiple filter paper-based pads to load enzymes and enzymatic substrates. The versatile analysis of different pesticides was carried by folding and unfolding the filter paper-based structure, without any addition of reagents and any sample treatment (i.e. dilution, filtration, pH adjustment). The paper-based platform was employed to detect paraoxon, 2,4-dichlorophenoxyacetic acid, and atrazine by exploiting the capability of these different types of pesticides (i.e. organophosphorus insecticides, phenoxy-acid herbicides, and triazine herbicide) to inhibit butyrylcholinesterase, alkaline phosphatase, and tyrosinase, respectively. The degree of inhibition correlating to the quantity of pesticides was evaluated by chronoamperometrically monitoring the enzymatic activity in the absence and in the presence of pesticides by using a portable potentiostat. To improve the sensitivity, the paper-based electrodes were modified with carbon black alone in the case of platforms for 2,4-dichlorophenoxyacetic acid and atrazine detection, or decorated with Prussian blue nanoparticles for the detection of paraoxon. The paper-based device was applied for the detection of paraoxon, 2,4-dichlorophenoxyacetic acid, and atrazine at ppb level in both standard solutions and river water sample. The accuracy of this origami multiple paper-based electrochemical biosensor was evaluated in river water by recovery studies, obtaining satisfactory values (e.g. for paraoxon 90 ±â€¯1% and 88 ±â€¯2%, for 10 and 20 ppb, respectively). The proposed three-dimensional origami paper device allows for rapid, cost-effective and accurate pesticide detection in surface water as a result of combining filter and office papers, screen-printing, wax-printing and nanomaterial technology.

Electrochemical immunosensor array using a 96-well screen-printed microplate for aflatoxin B1 detection.

A novel analytical immunosensor array, based on a microtiter plate coupled to a multichannel electrochemical detection (MED) system using the intermittent pulse amperometry (IPA) technique, is proposed for the detection of aflatoxin B1 (AFB1). In the present work, the electrochemical behaviour and electroanalytical performance of the thick-film carbon sensors (also designated as screen-printed electrodes) incorporated in the multichannel electrochemical plate were first evaluated. Then the 96-well screen-printed microplate was modified in accord with a competitive indirect enzyme-linked immunoassay (ELISA) format for aflatoxin B1 detection. The measurements were performed using both spectrophotometric and electrochemical procedures and the results of the calibration curves, detection limit (LOD), sensitivity and reproducibility of the respective assay systems were evaluated. The immunoassay was then applied for analysis of corn samples spiked with AFB1 before and after the extraction treatment, in order to study the extraction efficiency and the matrix effect, respectively. These studies have shown that using this system, AFB1 can be measured at a level of 30 pg/mL and with a working range between 0.05 and 2 ng/mL. Good recoveries (103+/-8%) were obtained, demonstrating the suitability of the proposed assay for accurate determination of the AFB1 concentration in corn samples. The specificity of the assay was assessed by studying the cross-reactivity of PAb relative to AFB1. The results indicated that the PAb could readily distinguish AFB1 from other aflatoxins, with the exception for AFG1.

ELIME assay vs Real-Time PCR and conventional culture method for an effective detection of Salmonella in fresh leafy green vegetables.

The detection of Salmonella according to EC regulation is still primarily based on traditional microbiological culture methods that may take several days to be completed. The purpose of this work is to demonstrate the applicability of an Enzyme-Linked-Immuno-Magnetic-Electrochemical (ELIME) assay, recently developed by our research group for the detection of salmonella in irrigation water, in fresh (raw and ready-to-eat) leafy green vegetables by comparison with Real-Time PCR (RTi-PCR) and ISO culture methods. Since vegetables represent a more complex matrix than irrigation water, preliminary experiments were carried out on two leafy green vegetables that resulted negative for salmonella by the ISO method. 25g of these samples were experimentally inoculated with 1-10 CFU of S. Napoli or S. Thompson and pre-enriched for 20h in two different broths. At this time aliquots were taken, concentrated at different levels by centrifugation, and analyzed by ELIME and RTi-PCR. Once selected the best culture medium for salmonella growth, and the optimal concentration factor suitable to reduce the sample matrix effect, enhancing the out-put signal, several raw and ready-to-eat leafy green vegetables were artificially inoculated and pre-enriched. Aliquots were then taken at different incubation times and analyzed with both techniques. Results obtained showed that 20 and 8h of pre-enrichment were required to allow the target salmonella (1-10 CFU/25g) to multiply until reaching a detectable concentration by ELIME and RTi-PCR assays, respectively. A confirmation with the ISO culture method was carried out. Based on the available literature, this is the first report of the application of an ELISA based method for the detection of Salmonella in vegetables.

Early echographical assessment of minimal lesions of cavernosum corpora and tunica albuginea in subjects with erectile dysfunction, suggestive of La Peyronie's disease.

The aim of the study is to evaluate the incidence and the echographic characteristics of minimal lesions of cavernosum corpora and tunica albuginea (TA) in subjects reporting erectile dysfunction (ED), which could suggest the suspicious of La Peyronie's disease (LPD). In total, 185 patients (pts) underwent dynamic penile Ultrasound Color Doppler (USCD) for ED. None of the pts presented any clinical symptoms or any clinical findings for LPD. In this study we evaluated, using USCD, thickness, echogenicity, regularity of the surface profile of the dorsal TA, the intercavernous and the intercaverno-spongeous septa, and the extension of the eventual pathologic lesions. In all, 16 pts (8.7%) presented minimal lesions at the ultrasound examinations. In nine of these pts (56%) the lesion was localized at the dorsal position, in six (38%) on the intercavernous septum and in one patient (6%) in both positions. The dorsal lesions were represented in nodular form in four pts (4%), and in diffuse form in five pts (55%). The nodular form was present in all the intercavernous septal lesions observed. As reported in the literature, USCD represents the investigative technique of choice in the study of LPD and in ED. Furthermore, the results of this study suggest that this technique could allow the localization of minimal lesions attributable to LPD during a preclinical phase of this disease. The localization of these lesions could permit to start a therapeutic approach during an early phase of the disease.