In vitro and in vivo characterization of the bifunctional µ and δ opioid receptor ligand UFP-505.

BACKGROUND AND PURPOSE: Targeting more than one opioid receptor type simultaneously may have analgesic advantages in reducing side-effects. We have evaluated the mixed µ opioid receptor agonist/ δ opioid receptor antagonist UFP-505 in vitro and in vivo. EXPERIMENTAL APPROACH: We measured receptor density and function in single µ, δ and µ /δ receptor double expression systems. GTPγ35 S binding, cAMP formation and arrestin recruitment were measured. Antinociceptive activity was measured in vivo using tail withdrawal and paw pressure tests following acute and chronic treatment. In some experiments, we collected tissues to measure receptor densities. KEY RESULTS: UFP-505 bound to µ receptors with full agonist activity and to δ receptors as a low efficacy partial agonist At µ, but not δ receptors, UFP-505 binding recruited arrestin. Unlike morphine, UFP-505 treatment internalized µ receptors and there was some evidence for internalization of δ receptors. Similar data were obtained in a µ /δ receptor double expression system. In rats, acute UFP-505 or morphine, injected intrathecally, was antinociceptive. In tissues harvested from these experiments, µ and δ receptor density was decreased after UFP-505 but not morphine treatment, in agreement with in vitro data. Both morphine and UFP-505 induced significant tolerance. CONCLUSIONS AND IMPLICATIONS: In this study, UFP-505 behaved as a full agonist at µ receptors with variable activity at δ receptors. This bifunctional compound was antinociceptive in rats after intrathecal administration. In this model, dual targeting provided no advantages in terms of tolerance liability. LINKED ARTICLES: This article is part of a themed section on Emerging Areas of Opioid Pharmacology. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.14/issuetoc.

Selective HCN1 block as a strategy to control oxaliplatin-induced neuropathy.

Chemotherapy-Induced Peripheral Neuropathy (CIPN) is the most frequent adverse effect of pharmacological cancer treatments. The occurrence of neuropathy prevents the administration of fully-effective drug regimen, affects negatively the quality of life of patients, and may lead to therapy discontinuation. CIPN is currently treated with anticonvulsants, antidepressants, opioids and non-opioid analgesics, all of which are flawed by insufficient anti-hyperalgesic efficacy or addictive potential. Understandably, developing new drugs targeting CIPN-specific pathogenic mechanisms would dramatically improve efficacy and tolerability of anti-neuropathic therapies. Neuropathies are associated to aberrant excitability of DRG neurons due to the alteration in the expression or function of a variety of ion channels. In this regard, Hyperpolarization-activated Cyclic Nucleotide-gated (HCN) channels are overexpressed in inflammatory and neuropathic pain states, and HCN blockers have been shown to reduce neuronal excitability and to ameliorate painful states in animal models. However, HCN channels are critical in cardiac action potential, and HCN blockers used so far in pre-clinical models do not discriminate between cardiac and non-cardiac HCN isoforms. In this work, we show an HCN current gain of function in DRG neurons from oxaliplatin-treated rats. Biochemically, we observed a downregulation of HCN2 expression and an upregulation of the HCN regulatory beta-subunit MirP1. Finally, we report the efficacy of the selective HCN1 inhibitor MEL57A in reducing hyperalgesia and allodynia in oxaliplatin-treated rats without cardiac effects. In conclusion, this study strengthens the evidence for a disease-specific role of HCN1 in CIPN, and proposes HCN1-selective inhibitors as new-generation pain medications with the desired efficacy and safety profile.

F1 Domain of the Leishmania (Leishmania) donovani Nucleoside Hydrolase Promotes a Th1 Response in Leishmania (Leishmania) infantum Cured Patients and in Asymptomatic Individuals Living in an Endemic Area of Leishmaniasis.

The Leishmania (Leishmania) donovani nucleoside hydrolase NH36 is the main antigen of the Leishmune® vaccine and one of the promising candidates for vaccination against visceral leishmaniasis. The antigenicity of the N-terminal (F1), the central (F2), or the C-terminal recombinant domain (F3) of NH36 was evaluated using peripheral blood mononuclear cells (PBMC) from individuals infected with L. (L.) infantum from an endemic area of visceral leishmaniasis of Spain. Both NH36 and F1 domains significantly increased the PBMC proliferation stimulation index of cured patients and infected asymptomatic individuals compared to healthy controls. Moreover, F1 induced a 19% higher proliferative response than NH36 in asymptomatic exposed subjects. In addition, in patients cured from visceral leishmaniasis, proliferation in response to NH36 and F1 was accompanied by a significant increase of IFN-γ and TNF-α secretion, which was 42-43% higher, in response to F1 than to NH36. The interleukin 17 (IL-17) secretion was stronger in asymptomatic subjects, in response to F1, as well as in cured cutaneous leishmaniasis after NH36 stimulation. While no IL-10 secretion was determined by F1, a granzyme B increase was detected in supernatants from cured patients after stimulation with either NH36 or F1. These data demonstrate that F1 is the domain of NH36 that induces a recall cellular response in individuals with acquired resistance to the infection by L. (L.) infantum. In addition, F1 and NH36 discriminated the IgG3 humoral response in patients with active visceral leishmaniasis due to L. (L.) donovani (Ethiopia) and L. (L.) infantum (Spain) from that of endemic and non-endemic area controls. NH36 showed higher reactivity with sera from L. (L.) donovani-infected individuals, indicating species specificity. We conclude that the F1 domain, previously characterized as an inducer of the Th1 and Th17 responses in cured/exposed patients infected with L. (L.) infantum chagasi, may also be involved in the generation of a protective response against L. (L.) infantum and represents a potential vaccine candidate for the control of human leishmaniasis alone, or in combination with other HLA epitopes/antigens.

Dietary cyanidin 3-glucoside from purple corn ameliorates doxorubicin-induced cardiotoxicity in mice.

BACKGROUND AND AIMS: Anthracyclines are effective anticancer drugs that have improved prognosis of hundred thousand cancer patients worldwide and are currently the most common chemotherapeutic agents used for the treatment of blood, breast, ovarian and lung cancers. However, their use is limited because of a cumulative dose-dependent and irreversible cardiotoxicity that can cause progressive cardiomyopathy and congestive heart failure. Aim of the present study was to determine the cardioprotective activity of a dietary source of cyanidin 3-glucoside (C3G), such as purple corn, against doxorubicin (DOX)-induced cardiotoxicity in mice. METHODS AND RESULTS: In vitro studies on murine HL-1 cardiomyocytes showed that pretreatment with both pure C3G and purple corn extract improved survival upon DOX treatment. However, C3G and purple corn extract did not affect the cytotoxic effect of DOX on human cancer cell lines. We then validated in vivo the protective role of a C3G-enriched diet against DOX-induced cardiotoxicity by comparing the effect of dietary consumption of corn isogenic lines with high levels of anthocyanins (purple corn - Red diet - RD) or without anthocyanins (yellow corn - Yellow diet - YD) incorporated in standard rodent diets. Results showed that mice fed RD survived longer than mice fed YD upon injection of a toxic amount of DOX. In addition, ultrastructural analysis of hearts from mice fed RD showed reduced histopathological alterations. CONCLUSION: Dietary intake of C3G from purple corn protects mice against DOX-induced cardiotoxicity.

Evaluation of enzymatic and non-enzymatic antioxidants in seminal plasma of men with genitourinary infections, varicocele and idiopathic infertility.

UNLABELLED: This study was aimed to assess the antioxidant enzymatic and non-enzymatic compounds in semen of infertile men. Seventy-four infertile patients were grouped according to their clinical diagnosis: genitourinary infection, varicocele, idiopathic infertility. Semen samples of fertile men represent the control. Semen characteristics were evaluated by light and transmission electron microscopy (TEM). TEM data was quantified with a mathematical formula, which provides numerical scores. Spectrophotometric and HPLC methods were used to measure the amount of reduced (GSH), oxidised glutathione (GSSG), ascorbic acid (AA) and malondialdehyde (MDA, marker of lipid peroxidation) and the activity of glutathione reductase, catalase (CAT), glutathione peroxidase. Infertile groups showed significantly decreased values of sperm parameters vs. CONTROLS: In infection and varicocele groups, the seminal MDA levels were significantly increased when compared to controls (p < 0.001), indicating an alteration of oxidative status and a peroxidative damage. In infection and varicocele groups, AA levels were reduced (p < 0.05) vs. control; in the varicocele group, the GSH levels were also decreased (p < 0.05). Significantly higher CAT activity was observed in infection and varicocele groups vs. fertile men (p < 0.001 and p < 0.05 respectively). The GSH/GSSG ratio was significantly decreased in varicocele and idiopathic infertility groups vs. control (p < 0.01). The study of the alteration of a single parameter of oxidative stress or of the antioxidant system may not have a relevant clinical value to estimate male fertilising potential and the background of infertility causes, since complex and multifactorial mechanisms are involved in different pathologies. In our study, each pathology is characterised by a definite pattern of markers such as MDA and enzymatic and non-enzymatic antioxidant compounds. In the different pathologies related to infertility, the identification of the complex of involved parameters could be useful in the diagnosis, prognosis and in the choice of a possible treatment such as specific antioxidant supplements.

Electronic tongue for microcystin screening in waters.

The potentiometric E-tongue system was employed for water toxicity estimation in terms of cyanobacterial microcystin toxins (MCs) detection. The data obtained from E-tongue were correlated to the MCs content detected by the standard chromatographic technique UHPLC-DAD (Ultra High Performance Liquid Chromatography with Diode Array Detector), as far as by the colorimetric enzymatic approach. The prediction of MCs released by toxic Microcystis aeruginosa strains was possible with Root Mean Squared Error of Validation (RMSEV) lower or very close to 1µg/L, the provisional guideline value of WHO for MCs content in potable waters. The application of E-tongue system opens up a new perspective offset for fast and inexpensive analysis in the field of environmental monitoring, offering also the possibility to distinguish toxin producing and non-toxic M. aeruginosa strains present in potable water.

A combined approach for predicting the cytotoxic effect of drug-nanoaggregates.

We present a combined spectroscopic and computational approach aimed to elucidate the mechanism of formation and activity of etoposide nanoaggregates upon release from dextran-etoposide conjugates. Etoposide is an anticancer drug that inhibits cell growth by blocking Topoisomerase II, the key enzyme involved in re-ligation of the DNA chains during the replication process. In silico and spectroscopic analysis indicate that released etoposide nanoaggregates have a different structure, stability, and bioactivity, which depend on the pH experienced during the release. Molecular dynamics simulation and in silico docking of etoposide dimers suggest that the aggregation phenomena inhibit etoposide bioactivity, yet without drastically preventing Topoisomerase II binding. We correlated the diminished cytotoxic activity exerted by dextran-etoposide conjugates on the A549 lung cancer cells, compared to the free drug, to the formation and stability of drug nanoaggregates.

Semen characteristics and malondialdehyde levels in men with different reproductive problems.

The aim of this study was to assess the level of malondialdehyde (MDA) in the seminal plasma of infertile men and to highlight a relationship between the level of MDA and semen parameters. Eighty-one infertile patients were divided into groups according to their clinical diagnosis: genitourinary infections, varicocele and idiopathic infertility. Semen quality was assessed by light and transmission electron microscopy (TEM). TEM data were quantified with a mathematical formula able to obtain a fertility index and the percentage of sperm apoptosis, immaturity, and necrosis. Seminal MDA levels were determined by spectrofluorometry. Scrotal Eco-color Doppler was used to detect the varicocele. Infected patients had a positive bacteriological semen analysis. A control group consisted of 14 normospermic fertile men. Fertile group showed significantly increased values of sperm concentration, motility, and fertility index compared to infertile groups. In the infertile groups, sperm motility, concentration, apoptosis, and fertility index were not significantly different. In infection group, the percentage of necrosis was significantly higher than that observed in fertile men, varicocele, and idiopathic infertility groups (p < 0.001). MDA levels increased significantly in infection group in comparison with varicocele group (p < 0.01), idiopathic infertility group, and fertile men (p < 0.001) and in varicocele group compared to idiopathic infertility group (p < 0.001). In infection group, MDA levels positively correlated with sperm concentration (p < 0.01), fertility index (p < 0.05), and necrosis (p < 0.001), whereas a negative correlation was found with motility (p < 0.01). In varicocele group MDA levels correlated positively with necrosis and negatively with immaturity (p < 0.05). In fertile men and idiopathic infertility group, they did not show any correlation. In conclusion, we suggest that the evaluation of seminal MDA may be a good marker for understanding pathologies responsible of a sperm motility reduction such as urogenital infections or inflammatory status.

Treatment of seriously compromised teeth: decision- making criteria and treatment options.

The aim of this article was to describe the case of a patient who presented to our attention with severe periodontal disease, complicated by furcation involvement on elements 16 and 17. In addition, the radiographic exam revealed the presence of a deep intrabony defect distal to tooth 15. Surgical therapy is performed after the resolution of the endodontic component of the intra-bony defect on the element 15 and consists on osteoplasty and ostectomy on 16, guided tissue regeneration (GTR) on 15, extraction of 17 and bi-laminar connective tissue graft for the coverage of the recession on tooth 13. The patient is visited monthly and 9 months after surgery, the definitive metal-ceramic crown is delivered and adapted to tooth 16. At 18 months, the patient's periodontal situation is re-evaluated and the pocket depth results healthy (probing depth of tooth 15=2 mm). The surgical practices reported in this work allowed for functional and esthetic rehabilitation of periodontally compromised teeth. The RSR and the GTR represent conservative surgery that allow the preservation of compromised dental elements and if properly performed, guarantee excellent survival rates of the elements in the arch. For these reasons, when it is possible, the RSR and the GTR are a valid alternative to implantology and are to be considered as the first therapeutic option in the treatment plan.

Retinopathy induced by drugs and herbal medicines.

Retina is the part of the eye suffering most damage from drugs. It is made up of a thin nervous membrane that covers the eye-ball internally, within the thickness of which three types of cells are ordered. In this paper we describe the drugs that are responsible for retinal side effects. Most commonly recognized drugs-induced retinopathy have a particular affinity for the retinal pigmented epithelium: antimalarials (quinine, hydroxychloroquine, mefloquine), phenothiazines, indomethacin, ethambutol, and desferrioxamine. Attention is especially focused on drugs more recently suspected of adverse reactions in the retina: vigabatrin, gabapentin, sildenafil, tamoxifen, isotretinoin, interferon, and omeprazole. Moreover, we referred some reports of retinopathy by herbal medicines and nutritional supplements (canthaxanthine, Gingko biloba L. and Glycyrrhiza glabra L.) This review is based on data published in scientific journals indexed by the PubMed and Medline databases. The last search of the literature was conducted in April 2008.

Non-contact ACL injuries in female athletes: an International Olympic Committee current concepts statement.

The incidence of anterior cruciate ligament (ACL) injury remains high in young athletes. Because female athletes have a much higher incidence of ACL injuries in sports such as basketball and team handball than male athletes, the IOC Medical Commission invited a multidisciplinary group of ACL expert clinicians and scientists to (1) review current evidence including data from the new Scandinavian ACL registries; (2) critically evaluate high-quality studies of injury mechanics; (3) consider the key elements of successful prevention programmes; (4) summarise clinical management including surgery and conservative management; and (5) identify areas for further research. Risk factors for female athletes suffering ACL injury include: (1) being in the preovulatory phase of the menstrual cycle compared with the postovulatory phase; (2) having decreased intercondylar notch width on plain radiography; and (3) developing increased knee abduction moment (a valgus intersegmental torque) during impact on landing. Well-designed injury prevention programmes reduce the risk of ACL for athletes, particularly women. These programmes attempt to alter dynamic loading of the tibiofemoral joint through neuromuscular and proprioceptive training. They emphasise proper landing and cutting techniques. This includes landing softly on the forefoot and rolling back to the rearfoot, engaging knee and hip flexion and, where possible, landing on two feet. Players are trained to avoid excessive dynamic valgus of the knee and to focus on the "knee over toe position" when cutting.

Collegiate rugby union injury patterns in New England: a prospective cohort study.

OBJECTIVE: To establish injury profile of collegiate rugby union in the USA. DESIGN/ SETTING: 31 men's and 38 women's collegiate rugby union teams prospectively recorded injuries during games and practice during the 2005-06 season. Three teams withdrew before data collection. An injury was defined as one: (1) occurring in an organised intercollegiate game or practice; and (2) requiring medical attention during or after the game or practice, or (3) resulting in any restriction of the athletes' participation for >or=1 day(s) beyond the day of injury, or in a dental injury. MAIN OUTCOME MEASURES: In total, 847 injuries (447 in men; 400 in women) during 48,026 practice (24,280 in men; 23,746 in women) and 25,808 game (13,943 in men; 11,865 in women) exposures were recorded. RESULTS: During games, injury rates of 22.5 (95% CI 20.2 to 25.0) and 22.7 (20.2 to 25.5) per 1000 game athletic exposures or 16.9 (15.1 to 18.9) and 17.1 (15.1 to 19.1) per 1000 player game hours were recorded for men and women, respectively. Over half of all match injuries were of major severity (>7 days' absence) (men 56%; women 51%) and the tackle was the game event most often associated with injury (men 48%; women 53%). CONCLUSIONS: Collegiate game injury rates for rugby were lower than rates recorded previously in men's professional club and international rugby and lower than reported by the National Collegiate Athletic Association Injury Surveillance System for American football, but similar to rates reported for men's and women's soccer in 2005-06.

Protective effect of silymarin on oxidative stress in rat brain.

Brain is susceptible to oxidative stress and it is associated with age-related brain dysfunction. Previously, we have pointed out a dramatic decrease of glutathione levels in the rat brain after acetaminophen (APAP) oral administration overdose. Silymarin (SM) is a mixture of bioactive flavonolignans isolated from Silybum marianum (L.) Gaertn., employed usually in the treatment of alcoholic liver disease and as anti-hepatotoxic agent in humans. In this study, we have evaluated the effect of SM on enzymatic and non enzymatic antioxidant defensive systems in rat brain after APAP-induced damage. Male albino Wistar rats were treated with SM (200 mg/kg/die orally) for three days, or with APAP single oral administration (3 g/kg) or with SM (200 mg/kg/die orally) for 3 days and APAP single oral administration (3 g/kg) at third day. Successively the following parameters were measured: reduced and oxidized glutathione (GSH and GSSG), ascorbic acid (AA), enzymatic activity variations of superoxide dismutase (SOD) and malondialdehyde levels (MDA). Our results showed a significant decrease of GSH levels, AA levels and SOD activity and an increase of MDA and GSSG levels after APAP administration. After SM administration GSH and AA significantly increase and SOD activity was significantly enhanced. In the SM+APAP group, GSH values significantly increase and the others parameters remained unchanged respect to control values. These results suggest that SM may to protect the SNC by oxidative damage for its ability to prevent lipid peroxidation and replenishing the GSH levels.

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.

Study of a toxin-alkaline phosphatase conjugate for the development of an immunosensor for tetrodotoxin determination.

This paper describes a direct competitive immunoenzymatic spectrophotometric assay (ELISA) for tetrodotoxin (TTX) determination and the adaptation of this method for use in an electrochemical assay format. The novelty of this work involves the use of the antigen labelled with alkaline phosphatase (AP); this conjugate was prepared in our laboratory as there is no commercially available conjugate of any kind for TTX. The new conjugate was characterized in terms of its affinity for the specific antibody as well as the residual concentration and the residual activity of the enzyme (AP) incorporated as label. The proposed method based on the new conjugate showed satisfactory results for TTX determination: for the spectrophotometric method the dynamic range was 4-15 ng mL(-1) with a limit of detection (LOD) of 2 ng mL(-1) (R=0.9247), whereas for the electrochemical protocol the dynamic range was 2-50 ng mL(-1) and the LOD was 1 ng mL(-1).

An electrochemical immunosensor for aflatoxin M1 determination in milk using screen-printed electrodes.

The production and assembling of disposable electrochemical AFM1 immunosensors, which can combine the high selectivity of immunoanalysis with the ease of the electrochemical probes, has been carried out. Firstly immunoassay parameters such as amounts of antibody and labelled antigen, buffer and pH, length of time and temperature of each steps (precoating, coating, binding and competition steps) were evaluated and optimised in order to set up a spectrophotometric enzyme-linked immunosorbent assay (ELISA) procedure. This assay exhibited a working range between 30 and 160 ppt in a direct competitive format. Then electrochemical immunosensors were fabricated by immobilising the antibodies directly on the surface of screen-printed electrodes (SPEs), and allowing the competition to occur between free AFM1 and that conjugated with peroxidase (HRP) enzyme. The electrochemical technique chosen was the chronoamperometry, performed at -100 mV. Furthermore, studies of interference and matrix effects have been performed to evaluate the suitability of the developed immunosensors for the analysis of aflatoxin M1 directly in milk. Results have shown that using screen-printed electrodes aflatoxin M1 can be measured with a detection limit of 25 ppt and with a working range between 30 and 160 ppt. A comparison between the spectrophotometric and electrochemical procedure showed that a better detection limit and shorter analysis time could be achieved using electrochemical detection.

T-cell engineering by a chimeric T-cell receptor with antibody-type specificity for the HIV-1 gp120.

Immune-based approaches of cell therapy against viral pathogens such as the human immunodeficiency virus type 1 (HIV-1) could be of primary importance for the control of this viral infection. Here, we designed a chimeric cell surface receptor (105TCR) to provide primary human T-lymphocytes with antibody-type specificity for the HIV-1 envelope glycoprotein. This receptor includes the single chain Fv domain of the neutralizing anti-gp120 human monoclonal antibody F105, CD8alpha hinge and the transmembrane and the cytoplasmic domains of TCRzeta. Our results show that 105TCR is expressed at the cellular surface and is capable of recognizing the HIV-1 envelope glycoprotein inducing highly efficient effector T-cell responses, including extracellular signal-regulated kinase phosphorylation and cytokine secretion. Moreover, human primary CD8+ T-lymphocytes transduced by oncoretroviral and lentiviral vectors containing the 105TCR gene are able to mediate in vitro-specific cytolysis of envelope-expressing cells and HIV-1-infected CD4+ T-lymphocytes. These findings suggest that 105TCR is particularly suited for in vivo efficacy studies.

Injuries in martial arts: a comparison of five styles.

OBJECTIVE: To compare five martial arts with respect to injury outcomes. METHODS: A one year retrospective cohort was studied using an injury survey. Data on 263 martial arts participants (Shotokan karate, n = 114; aikido, n = 47; tae kwon do, n = 49; kung fu, n = 39; tai chi, n = 14) were analysed. Predictor variables included age, sex, training frequency (3 h/week), experience (<3 years v >or=3 years), and martial art style. Outcome measures were injuries requiring time off from training, major injuries (>or=7 days off), multiple injuries (>or=3), body region, and type of injury. Logistic regression was used to determine odds ratios (OR) and confidence intervals (CI). Fisher's exact test was used for comparisons between styles, with a Bonferroni correction for multiple comparisons. RESULTS: The rate of injuries, expressed as percentage of participants sustaining an injury that required time off training a year, varied according to style: 59% tae kwon do, 51% aikido, 38% kung fu, 30% karate, and 14% tai chi. There was a threefold increased risk of injury and multiple injury in tae kwon do than karate (p<0.001). Subjects >or=18 years of age were at greater risk of injury than younger ones (p<0.05; OR 3.95; CI 1.48 to 9.52). Martial artists with at least three years experience were twice as likely to sustain injury than less experienced students (p<0.005; OR 2.46; CI 1.51 to 4.02). Training >3 h/week was also a significant predictor of injury (p<0.05; OR 1.85; CI 1.13 to 3.05). Compared with karate, the risks of head/neck injury, upper extremity injury, and soft tissue injury were all higher in aikido (p<0.005), and the risks of head/neck, groin, and upper and lower extremity injuries were higher in tae kwon do (p<0.001). No sex differences were found for any of the outcomes studied. CONCLUSIONS: There is a higher rate of injury in tae kwon do than Shotokan karate. Different martial arts have significantly different types and distribution of injuries. Martial arts appear to be safe for young athletes, particularly those at beginner or intermediate levels.

Disposable immunosensor for the determination of domoic acid in shellfish.

The construction of an electrochemical immunosensor coupled to differential pulse voltammetry (DPV) for the detection of domoic acid (DA), a neurotoxic aminoacid responsible for the human syndrome known as "Amnesic Shellfish Poisoning" (ASP), is proposed here. The method involves the use of disposable screen-printed electrodes (SPEs) for the immunosensor development based on a "competitive indirect test". Domoic acid conjugated to bovine serum albumin (BSA-DA) was coated onto the working electrode of the SPE, followed by incubation with sample (or standard toxin) and anti-DA antibody. An anti-goat IgG-alkaline phosphatase (AP) conjugate was used for signal generation. A spectrophotometric enzyme-linked immunosorbent assay (ELISA) was used in a preliminary phase of development, prior to transferring the assay to the SPEs. Results showed a detection limit equal to 5 ng/ml of toxin. The electrochemical system is simple and cost-effective due to the disposable nature of the SPEs, and the analysis time is 150 min, shorter than that for the spectrophotometric method. The suitability of the assay for DA quantification in mussels was also evaluated. Samples were spiked with DA before and after the sample treatment to study the extraction efficiency and the matrix effect, respectively. After treatment, samples were analysed using a 1:250 v/v dilution in PBS-M (phosphate saline buffer pH 7.4 + CH3OH 10%) to minimise the matrix effect and allow for the detection of 20 microg/g of DA in mussel tissue. This represents the maximum acceptable limit defined by the Food and Drug Administration [Compliance Programme 7303.842. Guidance Levels, Table 3, p. 248, http://www.fda.org]. The optimised ELISA systems were then used, in parallel with a conventional HPLC method, to detect and confirm DA in shellfish extract in order to verify the performance of the electrochemical system. Very good recoveries were obtained, demonstrating the suitability of the proposed assay for accurate determination of the DA concentration in mussel samples.