The Italian panorama of cannabis light preparation: Determination of cannabinoids by LC-UV.

Cannabis light preparations are products derived or containing dried female inflorescences of Cannabis sativa belonging to Chemotype III (THC/CBD ratio <<1); the total THC (THC+THCA) content in the crop must not exceed 0.2 % in accordance with the EU regulation. In Italy the most recent law for industrial hemp (242/2016) states that only for farmers this limit is extended to 0.6 %. On the other hand, the Ministry of the Interior published a note stating that the sale or the presence in the markets of products (inflorescences, concentrates, essences and resins) or plants with concentrations higher than 0.5 % constitutes a crime. In this confusing legislation framework, it is very important to assess the legality of hemp, determining the total amount of THC. To this end a reliable LC-UV analytical method was developed and validated taking into account parameters such as precision, accuracy, linearity, repeatability of peak area and retention time, limit of detection (LOD= 0.002 % for all cannabinoids) and limit of quantification (LOQ= 0.005 % for all cannabinoids). Accuracy was expressed as the relative error (Er%), while precision was measured as the coefficient of variation (CV%). A CV% below 3 % and Er% between ± 6 % were obtained. The linearity was proven in the concentration range 0.005-1 % for THC, THCA and CBN and 0.005 %-50 % for CBD and CBDA. The analytical method was applied to more than nine hundred cannabis light samples. Based on the law 242/2016, only 18 % of the crops are to be considered legal for the market (total THC<0.2 %). If the circular of the Ministry of the Interior should be converted as a proper law, a substantial amount of cannabis light preparations (24 %) would be considered illegal (total THC>0.5 %). On the other hand, the most of the inflorescences (58 %) have a total THC content comprised between 0.2 % and 0.5 %, and it is not clear whether these products could be sold or not. Moreover, Cannabis light products are not authorized for human consumption, even if everybody knows that this is their primary use. In conclusion, the cannabis light panorama in Italy is quite confused and more specific and clear legislation should be proposed.

Fast Detection of 10 Cannabinoids by RP-HPLC-UV Method in Cannabis sativa L.

Cannabis has regained much attention as a result of updated legislation authorizing many different uses and can be classified on the basis of the content of tetrahydrocannabinol (THC), a psychotropic substance for which there are legal limitations in many countries. For this purpose, accurate qualitative and quantitative determination is essential. The relationship between THC and cannabidiol (CBD) is also significant as the latter substance is endowed with many specific and non-psychoactive proprieties. For these reasons, it becomes increasingly important and urgent to utilize fast, easy, validated, and harmonized procedures for determination of cannabinoids. The procedure described herein allows rapid determination of 10 cannabinoids from the inflorescences of Cannabis sativa L. by extraction with organic solvents. Separation and subsequent detection are by RP-HPLC-UV. Quantification is performed by an external standard method through the construction of calibration curves using pure standard chromatographic reference compounds. The main cannabinoids dosed (g/100 g) in actual samples were cannabidiolic acid (CBDA), CBD, and Δ9-THC (Sample L11 CBDA 0.88 ± 0.04, CBD 0.48 ± 0.02, Δ9-THC 0.06 ± 0.00; Sample L5 CBDA 0.93 ± 0.06, CBD 0.45 ± 0.03, Δ9-THC 0.06 ± 0.00). The present validated RP-HPLC-UV method allows determination of the main cannabinoids in Cannabis sativa L. inflorescences and appropriate legal classification as hemp or drug-type.

A peroxiredoxin-based proteinaceous scaffold for the growth and differentiation of neuronal cells and tumour stem cells in the absence of prodifferentiation agents.

The use of nanoscale materials in the design of scaffolds for CNS tissue is increasing, due to their ability to promote cell adhesion, to mimic an extracellular matrix microenvironment and to interact with neuronal membranes. In this framework, one of the major challenges when using undifferentiated neural cells is how to control the differentiation process. Here we report the characterization of a scaffold based on the self-assembled nanotubes of a mutant of the protein peroxiredoxin (from Schistosoma mansoni or Bos taurus), which allows the growth and differentiation of a model neuronal cell line (SHSY5Y). The results obtained demonstrate that SHSY5Y cells grow without any sign of toxicity and develop a neuronal phenotype, as shown by the expression of neuronal differentiation markers, without the use of any differentiation supplement, even in the presence of serum. The prodifferentiation effect is demonstrated to be dependent on the formation of the protein nanotube, since a wild-type (WT) form of the peroxiredoxin from Schistosoma mansoni does not induce any differentiation. The protein scaffold was also able to induce the spread of glioblastoma cancer stem cells growing in neurospheres and allowing the acquisition of a neuron-like morphology, as well as of immature rat cortical neurons. This protein used here as coating agent may be suggested for the development of scaffolds for tissue regeneration or anti-tumour devices. Copyright © 2016 John Wiley & Sons, Ltd.

PDZ Domain in the Engineering and Production of a Saporin Chimeric Toxin as a Tool for targeting Cancer Cells.

In this paper we have studied a PDZ protein domain as a possible tool for cellular targeting of the ribosome inactivating protein Saporin, exploiting the ability of PDZ domains to recognize and bind short peptide sequences located at the C-terminus of a cognate protein. We have focused our attention on the PDZ domain from hCASK (Human calcium/calmodulin-dependent serine protein kinase) that binds extracellular CD98 in epithelial cells, being this antigen recognized as a marker for several human tumors and particularly considered a negative prognostic marker for human glioblastoma. We produced recombinant fusions of one or two hCASK-PDZ domains with the ribosome inactivating protein Saporin and assayed them on two human glioblastoma cell lines (GL15 and U87). These constructs proved to be toxic, with increasing activity as a function of the number of PDZ domains, and induce cell death by apoptotic mechanisms in a dose-dependent and/or time dependent manner.

Thoracic kyphosis associated with hemivertebra.

OBJECTIVE: To radiographically assess the influence of thoracic hemivertebra on kyphosis and neurologic status in 45 dogs. STUDY DESIGN: Case series. ANIMALS: French Bulldogs without neurologic signs (n = 41) and 4 dogs with compressive spinal cord disease associated with hemivertebra. METHODS: Thoracic spinal radiographs were divided into 3 groups: 1 = dogs with no hemivertebra, 2 = dogs with hemivertebra but no associated neurologic signs, and 3 = dogs with hemivertebra associated with clinical signs. Vertebral canal height, vertebral column angle, and the step between 2 adjacent vertebrae were measured in each group. RESULTS: Vertebral canal height was not significantly different in the 3 groups; however, mean vertebral column angle was significantly (P < .001) different between groups 1 (178°) and 2 (169°), and group 3 (133°). Median cranial step was significantly different between groups (P < .001): 0 mm (group 1), 0.3 mm (group 2), 3 mm (group 3). CONCLUSIONS: Vertebral canal height does not differ between normal dogs and dogs with thoracic hemivertebra. There is an important association between neurologic signs and kyphosis and subluxation. CLINICAL RELEVANCE: Vertebral realignment without vertebral canal opening could lead to spinal cord decompression in dogs affected by thoracic hemivertebra.

Preoperative immunosuppressive therapy and surgery as a treatment for anal furunculosis.

OBJECTIVES: To evaluate the efficacy of combining preoperative immunosuppressive therapy with surgical excision for treatment of anal furunculosis (AF) in dogs. STUDY DESIGN: Retrospective study. ANIMALS: Dogs (n=25) with stages 1-4 AF. METHODS: Preoperative immunosuppressive therapy was either cyclosporine A (CyA) alone or in combination with ketoconazole (Group 1; n=18), or azathioprine combined with prednisolone (Group 2; n=7). Surgical excision of residual draining tracts, cryptectomy, and anal sacculectomy were performed. Only dogs with postoperative clinical follow-up exceeding 9 months were retained for the study. RESULTS: Both immunosuppressive protocols were effective in reducing progression of AF. Subsequent draining tracts excision, cryptectomy, and anal sacculectomy were bilateral (12 dogs) or unilateral (13 dogs of which 4 had bilateral anal sacculectomy). Postoperative recovery was uneventful, except for 2 dogs that had wound breakdown. Recurrence was not observed in any of the dogs that had bilateral surgical excision and or in 9 dogs that had unilateral excision. CONCLUSION: Preoperative immunosuppressive therapy, combined with bilateral surgical resection of affected tissue consistently, resulted in resolution of AF. Four dogs that had recurrence had unilateral excision despite initial bilateral involvement, suggesting that all diseased tissue should be excised. CLINICAL RELEVANCE: These preliminary results suggest that immunosuppressive therapy before surgical excision for AF yields minimizes recurrence in dogs.

Automatic quantitative evaluation of emotions in E-learning applications.

The long term goal of our research is to develop a tool for recognizing human emotions during e-learning processes. This could be accomplished by combining quantitative indexes extracted from non-invasive recordings of four physiological signals: namely skin conductance, blood volume pulse, electrocardiogram and electroencephalogram. Wearable, non-invasive sensors, communicating with a PC, were applied to 30 students and data were collected during exposure to three different computer-mediated content stimuli designed to evoke specific emotional states: stress, relaxation and engagement. In this paper we describe both the general emotion evaluation algorithm, and present a preliminary results suggesting that some of the quantitative indexes may be successful in characterizing and distinguishing between the three different emotional states.