Toxicological analysis of pharmaceutical and illicit drugs in drug-facilitated sexual assault by LC-MS/MS using supramolecular biosolvents

The research carried out in this doctoral thesis focused on the development of an innovative and eco-friendly extraction method for the qualification and quantification of multi-class drugs involved in drug-facilitated sexual assaults (DFSA) from several biological matrices using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The extraction method was based on the use of supramolecular solvents (SUPRASs) and the target compounds included benzodiazepines, z-hypnotic drugs, amphetamine derivatives, cocaine and its metabolites and other miscellaneous compounds that are frequently used in DFSA. The developed green extraction method was applied to two biological matrices: liquid biological sample (urine) in chapter II and a solid biological sample (hair) in chapter III. In the Introduction, a general overview on the DFSA cases and involved compounds was reported, then the principle of green chemistry and the available extraction techniques used in forensic toxicology filed, and finally, the need of developing extraction method that allows the green analysis using SUPRASs was discussed. The results and discussion section starts with a review article (Chapter I) discussing the analysis of conventional and nonconventional forensic specimens in DFSA by LC-MS/MS. This review article provides a critical illustration about the DFSA in the last 10 years including the most common drugs reported in literature from different countries around the world and their prevalence. The review concluded that alcohol alone or in combination with a variety of pharmaceutical and illegal drugs (e.g., benzodiazepines, z-hypnotics drugs, cannabinoids, or cocaine) is still on top in the DFSA cases reported around the world. Moreover, the article reviewed the selection requirement of the appropriate biological samples and the increased use of the nonconventional samples (hair, vitreous humor, etc.) in such investigations. It is pointed out in the review that although liquid-liquid extraction (LLE) and solid-phase extraction (SPE) are the most often used extraction procedures, there is a growing interest in the development of microextraction formats and dilute and shoot tactics. Although GCMS is still employed in forensic routine analysis, LC-MS/MS (both low and highresolution MS) has been established as a more ideal alternative for multiple drug analysis due to its high sensitivity, specificity, and lack of the necessity for derivatization. Chapter II was performed to develop an innovative, green, and effective technique for extracting specific DFSA compounds from human urine using SUPRASs. The method has been optimized and validated for the extraction of 23 targeted-DFSA substances. The SUPRAS extraction process, which involves stirring, centrifugation, and dilution, is simple, quick, and environmentally friendly. The extracted SUPRAS samples are then analyzed using LC-MS/MS. The study demonstrates that the proposed extraction method has a high extraction efficiency ranging from 79% to 119% for many target compounds. Matrix effects are found to be tolerable for most compounds. Additionally, the method achieves low detection and quantification limits, surpassing the minimal performance requirements for these compounds. These findings indicate that the SUPRAS-based extraction technique holds great promise for monitoring illicit substances in DFSA cases within forensic laboratories. After investigating the SUPRAS extraction technique to liquid biological samples (urine), the efficiency of SUPRAS method was planned to be studied on solid biological samples (hair). For this reason, the third section (Chapter III) discusses the development and validation of a single-step extraction method for analysing DFSA compounds in hair samples. Overall, the SUPRAS-based method offers several advantages, including its green and eco-friendly nature, simplified extraction process, increased sensitivity, broad compound coverage, high extraction efficiency, and practical applicability in real forensic sexual assault cases. At the end of the thesis, the most relevant conclusions of the chapters are pointed out in the Conclusion section. Finally, in the Annexes, the informed consent for extraction, use and storage of biological samples, as well as the communications presented at scientific conferences, are included.La investigación llevada a cabo en esta tesis doctoral se centró en el desarrollo de un método de extracción innovador y respetuoso con el medio ambiente para la identificación y cuantificación de fármacos de múltiples clases involucrados en agresiones sexuales facilitadas por drogas (DFSA) a partir de diversas matrices biológicas, utilizando cromatografía líquida-espectrometría de masas en tándem (LCMS/ MS). El método de extracción se basó en el uso de disolventes supramoleculares (supramolecular solvents, SUPRASs) y las drogas seleccionadas incluyeron benzodiacepinas, fármacos hipnóticos tipo Z, derivados de anfetaminas, cocaína y sus metabolitos, y otros compuestos diversos que se utilizan con frecuencia en casos de DFSA. El método de extracción desarrollado se aplicó a dos matrices biológicas: muestra biológica líquida (orina) en el capítulo II y muestra biológica sólida (pelo) en el capítulo III. En la Introducción se presenta una visión general de los casos de DFSA y los compuestos involucrados, a continuación, se aborda el principio de la química verde y las técnicas de extracción disponibles utilizadas en el campo de la toxicología forense, y finalmente se discute la necesidad de desarrollar métodos de extracción más sostenibles. La sección de resultados y discusión comienza con un artículo de revisión (Capítulo I), que trata sobre el análisis de muestras forenses convencionales y no convencionales en casos de DFSA mediante LC-MS/MS. Este artículo de revisión proporciona una revisión crítica sobre los casos de DFSA en los últimos 10 años, incluyendo los fármacos más comunes reportados en la literatura de diferentes países de todo el mundo y su prevalencia. La revisión concluyó que el alcohol solo o en combinación con una variedad de fármacos farmacéuticos e ilegales (por ejemplo, benzodiacepinas, fármacos hipnóticos tipo Z, cannabinoides o cocaína) sigue siendo el más común en los casos de DFSA reportados en todo el mundo. Además, el artículo revisó los requisitos de selección de la muestra biológica apropiada en DFSA y el aumento en el uso de muestras no convencionales (pelo, humor vítreo, etc.) en tales investigaciones. Se señala en la revisión que, aunque la extracción líquido-líquido (LLE) y la extracción en fase sólida (SPE) son los procedimientos de extracción más utilizados, existe un creciente interés en el desarrollo de formatos de microextracción y tácticas. de dilución y análisis directo. Aunque la GC-MS todavía se utiliza en el análisis forense de rutina, la LC-MS/MS (tanto en MS de baja como de alta resolución) se ha establecido como una alternativa más ideal para el análisis de múltiples fármacos debido a su alta sensibilidad, especificidad y la falta de necesidad de derivatización. En el Capítulo II se desarrolló una técnica innovadora, ecológica y efectiva para extraer compuestos específicos de DFSA de la orina humana utilizando SUPRASs. El método ha sido optimizado y validado para la extracción de 23 sustancias específicas de DFSA. El proceso de extracción con SUPRASs, que incluye agitación, centrifugación y dilución, es simple, rápido y respetuoso con el medio ambiente. Las muestras de SUPRAS extraídas se analizan directamente utilizando LC-MS/MS. El estudio demuestra que el método de extracción propuesto tiene una alta eficiencia de extracción que oscila entre el 79% y el 119% para muchos compuestos objetivo. Los efectos de matriz se consideran tolerables para la mayoría de los compuestos. Además, el método alcanza límites de detección y cuantificación bajos, superando los requisitos mínimos de rendimiento para estos compuestos. Estos hallazgos indican que la técnica de extracción basada en SUPRAS es eficaz y respetuosa con el medio ambiente y tiene un gran potencial para detectar y cuantificar sustancias ilícitas en casos de DFSA en laboratorios forenses. Después de investigar el uso de la técnica de extracción SUPRAS en muestras biológicas líquidas (orina), se planeó estudiar la eficiencia del método SUPRAS en muestras biológicas sólidas (pelo). Por esta razón, la tercera sección (Capítulo III) trata sobre el desarrollo y la validación de un método de extracción para analizar compuestos de DFSA en muestras de pelo que consta de una única etapa. En general, el método basado en SUPRAS ofrece varias ventajas, incluyendo su naturaleza ecológica, proceso de extracción simplificado, mayor sensibilidad, amplia cobertura de compuestos, alta eficiencia de extracción y aplicabilidad en casos forenses reales de agresión sexual. Al final de la tesis, se destacan las conclusiones más relevantes de las investigaciones presentadas en los diferentes capítulos en la sección de Conclusiones. Finalmente, en los anexos, se incluyen el consentimiento informado para la extracción, uso y almacenamiento de muestras biológicas, así como las comunicaciones presentadas en conferencias científicas

Photoionization-ion mobility spectrometer for non-targeted screening analysis or for targeted analysis coupling a Tenax TA column

Photoionization-ion mobility spectrometer (UV-IMS) can be used as a screening instrument to distinguish gasoline vapors from other similar samples, obtaining characteristic fingerprints in a quick and simple analysis. A non-targeted approach can be used to resolve many problems, moreover, this device can also be used for targeted analysis. In this case a Tenax TA trap was connected to the UV-IMS as a pre-separation step in order to identify compounds present in the gasoline gaseous sample, such as benzene and toluene. Firstly, target analytes were trapped in a Tenax TA column at ambient temperature and then they were desorbed thermally in an oven and connected to the UV-IMS for their analysis. Instrumental parameters such as temperature ramp, sample gas flow rate, drift gas flow rate, adsorption time and adsorption flow rate were optimized to obtain high sensitivity, selectivity and precision to determine benzene and toluene in vapour gaseous samples. The UV-IMS was calibrated with standard gases generated in an exponential dilution flask, obtaining a limit of quantification of 1.8 and 1.9 mg m-3 for benzene and toluene, respectively. The method developed was successfully applied to identify these analytes in air samples close to a gasoline spillag

In ovarian cancer the prognostic influence of HER2/neu is not dependent on the CXCR4/SDF-1 signalling pathway

HER2/neu overexpression is a driving force in the carcinogenesis of several human cancers. In breast cancer the prognostic influence of HER2/neu was shown to be at least partly based on increased metastatic potential mediated by the chemokine–chemokine receptor pair SDF-1(CXCL12)/CXCR4. We wanted to evaluate the influence of HER2/neu on ovarian cancer prognosis and to investigate whether compromised survival would correlate with CXCR4 expression and/or SDF-1 abundance. Therefore, we analysed HER2/neu, CXCR4, and SDF-1 in 148 ovarian tumour samples by means of immunohistochemistry on tissue microarrays. Overexpression of HER2/neu was found in 27.6% of ovarian cancer tissues and in 15% of ovarian borderline tumours. In ovarian cancer patients, overexpression of HER2/neu correlated closely with overall survival (univariate hazard ratio (HR) 2.59, P=0.005; multiple corrected HR 1.92, P=0.074). In contrast, CXCR4 expression and SDF-1 abundance had no impact on overall survival, and both parameters were not correlated with HER2/neu expression. As expected, cytoplasmic CXCR4 expression and SDF-1 abundance correlated closely (P<0.0001). Our results confirm a univariate influence of HER2/neu expression on overall survival, which was completely independent of the expression of CXCR4 and the abundance of SDF-1, implying significant differences between the HER2/neu downstream pathways in ovarian cancer compared with breast cancer

Frequent downregulation of 14-3-3 σ protein and hypermethylation of 14-3-3 σ gene in salivary gland adenoid cystic carcinoma

14-3-3 σ, a target gene of the p53 tumour suppressor protein, has been shown to regulate the cell cycle at the G2/M checkpoint. Recent studies have demonstrated that 14-3-3 σ is downregulated by hypermethylation of the CpG island in several types of cancer. In this study, we investigated the expression and methylation status of 14-3-3 σ in human salivary gland adenoid cystic carcinoma (ACC) and mucoepidermoid carcinoma (MEC). Immunohistochemical analysis revealed that the positive expression rate of 14-3-3 σ in ACC (one out of 14) was markedly lower than that in MEC (ten out of 10). Since most of the ACCs carried the wild-type p53 protein, downregulation of 14-3-3 σ in ACC may not be due to the dysfunction of p53 pathway. Microdissection–methylation-specific PCR revealed that frequent hypermethylation of the 14-3-3 σ gene was observed in ACC when compared to that in MEC. In cultured-ACC cells, we confirmed the downregulation of 14-3-3 σ via hemimethylation of the gene by sequencing analysis after sodium bisulphite treatment. Furthermore, re-expression of 14-3-3 σ in the ACC cells was induced by the treatment with DNA demethylating agent, 5-aza-2′-deoxycytidine. Irradiation apparently induced the enhanced expression of 14-3-3 σ and G2/M arrest in normal salivary gland cells; however, in the ACC cells, neither induction of 14-3-3 σ nor G2/M arrest was induced by irradiation. These results suggest that downregulation of 14-3-3 σ might play critical roles in the neoplastic development and radiosensitivity of ACC

Cellular delivery of small interfering RNA by a non-covalently attached cell-penetrating peptide: quantitative analysis of uptake and biological effect

Cell-penetrating peptides (CPPs) have evolved as promising new tools to deliver nucleic acids into cells. So far, the majority of these delivery systems require a covalent linkage between carrier and cargo. To exploit the higher flexibility of a non-covalent strategy, we focused on the characterisation of a novel carrier peptide termed MPGα, which spontaneously forms complexes with nucleic acids. Using a luciferase-targeted small interfering RNA (siRNA) as cargo, we optimised the conditions for MPGα-mediated transfection of mammalian cells. In this system, reporter gene activity could be inhibited up to 90% with an IC(50) value in the sub-nanomolar range. As a key issue, we addressed the cellular uptake mechanism of MPGα/siRNA complexes applying various approaches. First, transfection of HeLa cells with MPGα/siRNA complexes in the presence of several inhibitors of endocytosis showed a significant reduction of the RNA interference (RNAi) effect. Second, confocal laser microscopy revealed a punctual intracellular pattern rather than a diffuse distribution of fluorescently labelled RNA-cargo. These data provide strong evidence of an endocytotic pathway contributing significantly to the uptake of MPGα/siRNA complexes. Finally, we quantified the intracellular number of siRNA molecules after MPGα-mediated transfection. The amount of siRNA required to induce half maximal RNAi was 10 000 molecules per cell. Together, the combination of methods provided allows for a detailed side by side quantitative analysis of cargo internalisation and related biological effects. Thus, the overall efficiency of a given delivery technique as well as the mechanism of uptake can be assessed

Analysis of chemokine and chemokine receptor expression in squamous cell carcinoma of the head and neck (SCCHN) cell lines

The purpose of this work was to analyze chemokine and chemokine receptor expression in untreated and in irradiated squamous cell carcinoma of the head and neck (SCCHN) tumor cell lines, aiming at the establishment of assays to test for the relevance of chemokine and chemokine receptor expression in the response of SCCHN to radiotherapy and radiochemotherapy. Five low passage and 10 established SCCHN lines, as well as two normal cell lines, were irradiated at 2 Gy or sham-irradiated, and harvested between 1 and 48 h after treatment. For chemokines with CC and CXC structural motifs and their receptors, transcript levels of target and reference genes were quantified relatively by real-time PCR. In addition, CXCL1 and CXCL12 protein expression was analyzed by ELISA. A substantial variation in chemokine and chemokine receptor expression between SCCHN was detected. Practically, all cell lines expressed CCL5 and CCL20, while CCL2 was expressed in normal cells and in some of the tumor cell lines. CXCL1, CXCL2, CXCL3, CXCL10, and CXCL11 were expressed in the vast majority of the cell lines, while the expression of CXCL9 and CXCL12 was restricted to fibroblasts and few tumor cell lines. None of the analyzed cell lines expressed the chemokines CCL3, CCL4, or CCL19. Of the receptors, transcript expression of CCR1, CCR2, CCR3, CCR5, CCR7, CCXR2, and CCXR3 was not detected, and CCR6, CXCR1, and CXCR4 expression was restricted to few tumor cells. Radiation caused up- and down-regulation with respect to chemokine expressions, while for chemokine receptor expressions down-regulations were prevailing. CXCL1 and CXCL12 protein expression corresponded well with the mRNA expression. We conclude that the substantial variation in chemokine and chemokine receptor expression between SCCHN offer opportunities for the establishment of assays to test for the relevance of chemokine and chemokine receptor expression in the response of SCCHN to radiotherapy and radiochemotherapy

Challenges of Implementing Formative Assessment

Formative assessment (FA) is an essential element of EFL classrooms. It allows instructors to amend, adjust, and monitor the process of teaching to reach their desired goals. However, this form of assessment is often overlooked in Iraqi EFL classrooms. As such, this study aims to investigate the challenges of implementing the FA strategies by Iraqi EFL university instructors and their attitudes to improve its use effectively in teaching. The data for this study were gathered through the observation of six EFL classes and semi-structured interviews conducted with three EFL instructors from three different Iraqi universities. The results revealed that there are many challenges for FA strategies implementation. Some of them were related to the EFL instructors’ insufficient knowledge to conduct FA strategies effectively and also their inability to create assessment criteria for their tasks and activities. Moreover, the time-consuming nature of the FA strategies and time limit of the class sessions makes it impossible for the instructors to integrate these strategies into their teaching. Furthermore, the instructors also reported that their students do not have enough knowledge of FA strategies, and cannot be involved in their implementation. Finally, the study recommends that training is needed for the Iraqi EFL instructors especially those with less experience on how to integrate and use FA strategies in the classroom.</jats:p