Performance characteristics of an ELISA screening assay for urinary synthetic cannabinoids

Synthetic cannabinoids are marketed as legal alternatives to cannabis, as routine urine cannabinoid immunoassays do not detect synthetic cannabinoids. Laboratories are challenged to identify these new designer drugs that are widely available and represent a major public health and safety problem. Immunoassay testing offers rapid separation of presumptive positive and negative specimens, prior to more costly and time‐consuming chromatographic confirmation. The Neogen SPICE ELISA kit targets JWH‐018 N‐pentanoic acid as a marker for urinary synthetic cannabinoids. Assay performance was evaluated by analyzing 2469 authentic urine samples with the Neogen immunoassay and liquid chromatography‐tandem mass spectrometry (LC‐MS/MS). Two immunoassay cut‐off concentrations, 5 and 10 µg/L, classified samples as presumptive positive or negative, followed by qualitative LC‐MS/MS confirmation for 29 synthetic cannabinoids markers with limits of detection of 0.5–10 µg/L to determine the assay's sensitivity, specificity and efficacy. Challenges at ±25% of each cut‐off also were investigated to determine performance around the cut‐off and intra‐ and inter‐plate imprecision. The immunoassay was linear from 1 to 250 µg/L (r² = 0.992) with intra‐ and inter‐plate imprecision of ≤5.3% and <9%, respectively. Sensitivity, specificity, and efficiency results with the 5 µg/L cut‐off were 79.9%, 99.7%, and 97.4% and with the 10 µg/L cut‐off 69.3%, 99.8%, and 96.3%, respectively. Cross‐reactivity was shown for 18 of 73 synthetic cannabinoids markers evaluated. Good sensitivity, specificity, and efficiency, lack of sample preparation requirements, and rapid semi‐automation documented that the Neogen SPICE ELISA kit is a viable method for screening synthetic cannabinoids in urine targeting JWH‐018 N‐pentanoic acid. Copyright © 2014 John Wiley & Sons, Ltd.     

Central nervous mechanisms of cough

Cough is an airway defensive reflex substantially consisting in a modified respiratory act. Transection experiments have shown that the fundamental structures responsible for this reflex are located within the medulla oblongata. Electrical stimulation applied to the medulla failed to provide convincing evidence of a cough centre distinct from the brainstem respiratory network. In fact, electrical stimuli affect not only neuronal somata, but also intramedullary cough-related pathways. Studies on the behaviour of medullary respiratory neurones have led to the conclusion that the same respiratory neurones involved in the generation of the eupnoeic pattern of breathing also participate in the production of the cough motor pattern. These findings support the existence of multifunctional neural networks in the mammal brainstem. B?tzinger complex expiratory neurones with augmenting discharge patterns have been suggested to convey an excitatory drive to the expiratory bulbospinal neurones of the caudal ventral respiratory group and, hence, to expiratory motoneurones. The excitatory drive to caudal medullary expiratory neurones is mediated by ionotropic glutamate receptors. Recent lines of evidence indicate that the B?tzinger complex and the caudal ventral respiratory group have a crucial role in determining both the inspiratory and the expiratory components of the cough motor pattern.     

Enhancer blocking activity located near the 3′ end of the sea urchin early H2A histone gene

The sea urchin early histone repeating unit contains one copy of each of the five histone genes whose coordinate expression during development is regulated by gene-specific elements. To learn how within the histone repeating unit a gene-specific activator can be prevented to communicate with the heterologous promoters, we searched for domain boundaries by using the enhancer blocking assay. We focused on the region near the 3′ end of the H2A gene where stage-specific nuclease cleavage sites appear upon silencing of the early histone genes. We demonstrated that a DNA fragment of 265 bp in length, defined as sns (for silencing nucleoprotein structure), blocked the enhancer activity of the H2A modulator in microinjected sea urchin embryos only when placed between the enhancer elements and the promoter. We also found that sns silenced the modulator elements even when placed at 2.7 kb from the promoter. By contrast, the enhancer activity of the modulator sequences, located downstream to the coding region, was not affected when sns was positioned in close proximity to the promoter. Finally, the H2A sns fragment placed between the simian virus 40 regulative region and the tk promoter repressed chloramphenicol acetyltransferase expression in transfected human cell lines. We conclude that 3′ end of the H2A gene contains sequence elements that behave as functional barriers of enhancer function in the enhancer blocking assay. Furthermore, our results also indicate that the enhancer blocking function of sns lacks enhancer and species specificity and that it can act in transient assays.     

Robot-assisted surgery for the radical treatment of deep infiltrating endometriosis with colorectal involvement: short- and mid-term surgical and functional outcomes

Purpose

Sexual and urinary dysfunctions are complications in radical treatment of deep infiltrating endometriosis (DIE) with colorectal involvement. The aim of this article is to report the preliminary results of our single-institution experience with robotic treatment of DIE, evaluating intraoperative and postoperative surgical outcomes and focusing on the impact of this surgical approach on autonomic functions such as urogenital preservation and sexual well-being.

Methods

From January 2011 through December 2013, a case series of 10 patients underwent robotic radical treatment of DIE with colorectal resection using the da Vinci System. Surgical data were evaluated, together with perioperative urinary and sexual function as assessed by means of self-administered validated questionnaires.

Results

None of the patients reported significant postoperative complications. Questionnaires concerning sexual well-being, urinary function, and impact of symptoms on quality of life demonstrated a slight worsening of all parameters 1 month after surgery, while data were comparable to the preoperative period 1 year after surgery. Dyspareunia was the only exception, as it was significantly improved 12 months after surgery.

Conclusions

Robot-assisted surgery seems to be advantageous in highly complicated procedures where extensive dissection and proper anatomy re-establishment is required, as in DIE with colorectal involvement. Our preliminary results show that robot-assisted surgery could be associated with a low risk of complications and provide good preservation of urinary function and sexual well-being.

     

Transferrin receptor 1 (TfR1) and putative stimulator of Fe transport (SFT) expression in iron deficiency and overload: an overview

Transferrin Receptor 1 (TfR1) and putative Stimulator of Fe Transport (SFT) represent two different proteins involved in iron metabolism in mammalian cells. The expression of TfR1 in the duodenum of subjects with normal body iron stores has been mainly localized in the basolateral portion of the cytoplasm of crypt cells, supporting the idea that this molecule may be involved in the sensing of body iron stores. In iron deficiency anemia TfR1 expression demonstrated an inverse relationship with body iron stores as assessed by immunohistochemistry with anti-TfR1 antibodies. In iron overload, TfR1 expression in the duodenum differed according to the presence or absence of the C282Y mutation in the HFE gene, being increased in HFE-related hemochromatosis and similar to controls in non-HFE-related iron overload. SFT is characterized by its ability to increase iron transport both through the transferrin dependent and independent uptake, and could thus affect iron absorption in the intestine. Immunohistochemistry using anti-SFT antibodies which recognize a putative stimulator of Fe transport of approximately 80 KDa revealed a localization of this protein in the apical part of the cytoplasm of enterocytes localized at the tip of the villi. The expression of the protein recognized by these antibodies was increased in iron deficiency, as well as in patients carrying the C282Y HFE mutation. Thus, the increased expression of both proteins only in patients with HFE-related hemochromatosis suggests that other factors should be involved in determining non-HFE-related iron overload.     

Contactless magnetocardiographic study of ventricular repolarization in intact Wistar rats: Evidence of gender-related differences

Abstract.Introduction: Magnetocardiographic (MCG) mapping is increasingly used for the non-invasive study of coronary artery disease (CAD), cardiomyopathy (CMP) and arrhythmias. MCG study of small animals with ischemia or CMP would provide useful experimental models for the interpretation of abnormal human patterns. The aim of this study was to assess the range of normality of MCG ventricular repolarization parameters, in intact adult rats.Methods and results: 10 adult Wistar rats (weight 250 – 350 grams) were investigated with a 36-channel MCG mapping instrumentation (sensitivity is 20 fT/ Hz, at 1 Hz), designed for clinical recordings in unshielded hospital laboratories. To assess ventricular repolarization, MCG Q-T_peak, Q-T_end, J-T_peak, J-T_end, T_peak-T_end intervals were measured. MCG imaging was obtained by automatic calculation of isofield contour maps, and with inverse source localization based on the Equivalent Current Dipole and Effective Magnetic Dipole models, in a semi-infinite space with homogeneous conductance. Magnetic field (MF) orientation, dynamics and stability during the J-T_end segment were also calculated. After averaging 300 s of continuous MCG recording, the S/N ratio was good enough for reproducible reconstruction of both atrial and ventricular magnetic maps and for three-dimensional localization of the underlying cardiac generators. Clear gender-related differences in ventricular repolarization duration were found, evidenced by significantly longer Q-T_end, J-T_end and T_peak-T_end intervals in females. The differences in MF orientation, and stability during the J-T_end segment were not significant. For J-T_peak MF dynamics, only the ratio between the strengths of the positive and negative poles was significantly lower in the female group (p < 0.05).Conclusions: Contactless MCG recording is a novel approach, which simplifies non-invasive mapping in small animals. In normal rats, gender-related differences of ventricular repolarization parameters equivalent to those reported in previous ECG studies were demonstrated.     

Ticagrelor and tirofiban in pregnancy and delivery: beyond labels

Journal of Thrombosis and Thrombolysis - Pregnancy-associated acute myocardial infarction is a rare condition usually occurring during the third trimester of pregnancy, and associated with...