Quantification of 108 illicit drugs and metabolites in bile matrix by LC–MS/MS for the toxicological testing of sudden death cases

Sudden death could occur after assumption of illicit drugs for recreational purposes in adults or after intoxication in children, and toxicological testing would help identify the cause of the death. Analytical methods sensitive and specific for the quantification of a great number of drugs and metabolites in at least 2 matrices should be used. Bile, collected postmortem, may be considered a specimen alternative to blood and urine to perform toxicological testing because of its extended detection window. The present study proposed a LC–MS/MS method to quantify 108 drugs and metabolites in bile. Compounds belonging to the drugs of abuse classes of amphetamines, benzodiazepines, cocaine derivatives, barbiturates, opioids, z-drugs, and psychedelics were analyzed. The sample preparation is simple and does not require solid-phase extraction. The proposed method showed an appropriate selectivity, specificity, accuracy, and precision of the calibrators and quality controls tested (precision < 15%; accuracy < 100 ± 15%). The sensitivity allowed to identify low amounts of drugs (e.g., morphine limit of detection = 0.2 μg/L; limit of quantification = 1.1 μg/L). There is no significant matrix effect, except for buprenorphine and 11-Nor-9-carboxy-Δ9-tetrahydrocannabinol. Carry-over was not present. Analytes were stable at least for 1 month at − 20 °C. Analyzing 13 postmortem specimens, methadone (50%), and cocaine (37.5%) resulted to be the most prevalent consumed substances; the concentrations quantified in bile resulted to be higher than the ones in blood suggesting bile as a potential new matrix for identifying illicit drugs and their metabolites

Pathology Laboratory Archives: Conservation Quality of Nucleic Acids and Proteins for NSCLC Molecular Testing

In the molecular era, proper archival conditions within pathology laboratories are crucial, especially for formalin-fixed paraffin-embedded (FFPE) tissue specimens retrieved years after the original diagnosis. Indeed, improper preservation can impact the integrity of nucleic acids and protein antigens. This study evaluates the quality status of stored FFPE blocks using multilevel omics approaches. FFPE blocks from 45 Non-Small Cell Lung Carcinoma (NSCLC) cases were analyzed. The blocks were collected from six different pathology archives across Italy with distinct environmental characteristics. Nucleic acids’ quantity and quality, as well as protein antigens, were assessed using various techniques, including MALDI-MSI. RNA was quantitatively higher, but more fragmented, compared to DNA. DNA quantity and quality were suitable for molecular analyses in 94.4% and 62.3% of samples, respectively. RNA quantity was adequate across all samples, but it was optimal only in 22.3% of cases. DNA quality started to deteriorate after 6–8 years, whereas RNA quality diminished only after 10 years of storage. These data might suggest a particular DNA susceptibility to FFPE blocks conservation. Immunohistochemical intensity decreased significantly after 6–8 years of storage, and MALDI-MSI analysis revealed that younger tissue blocks contained more unique proteomic signals than the older ones. This study emphasizes the importance of proper FFPE archiving conditions for molecular analyses. Governance should prioritize attention to pathology archives to ensure quality preservation and optimize predictive testing. By elucidating the nuances of FFPE block storage, this research paves the way for enhanced molecular diagnostics and therapeutic insights regarding oncology and beyond

Knowledge about tuberculosis among undergraduate health care students in 15 Italian universities: a cross-sectional study

Background: The Italian Study Group on Hospital Hygiene of the Italian Society of Hygiene, Preventive Medicine and Public Health conducted a multicentre survey aiming to evaluate undergraduate health care students' knowledge of tuberculosis and tuberculosis control measures in Italy. Methods. In October 2012-June 2013, a sample of medical and nursing students from 15 Italian universities were enrolled on a voluntary basis and asked to complete an anonymous questionnaire investigating both general knowledge of tuberculosis (aetiology, clinical presentation, outcome, screening methods) and personal experiences and practices related to tuberculosis prevention. Data were analysed through multivariable regression using Stata software. Results: The sample consisted of 2,220 students in nursing (72.6%) and medicine (27.4%) courses. Our findings clearly showed that medical students had a better knowledge of tuberculosis than did nursing students.Although the vast majority of the sample (up to 95%) answered questions about tuberculosis aetiology correctly, only 60% of the students gave the correct responses regarding clinical aspects and vaccine details. Overall, 66.9% of the students had been screened for tuberculosis, but less than 20% of those with a negative result on the tuberculin skin test were vaccinated. Multivariable regression analysis showed that age and type of study programme (nursing vs. medical course) were determinants of answering the questions correctly. Conclusions: Although our data showed sufficient knowledge on tuberculosis, this survey underlines the considerable need for improvement in knowledge about the disease, especially among nursing students. In light of the scientific recommendations concerning tuberculosis knowledge among students, progress of current health care curricula aimed to develop students' skills in this field is needed

Apoptosis en el cáncer de páncreas: ¿el beso de Judas?

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de Lectura: 27-05-2022El cáncer de páncreas (PDAC) conforma una patología de difícil diagnóstico, sobre todo sus metástasis, y con un tratamiento poco eficaz dada la resistencia intrínseca a los tratamientos quimioterápicos. Los macrófagos, especialmente los TAM (tumor associated macrophage), son muy abundantes en este cáncer. En esta tesis hemos hipotetizado que son responsables tanto de la producción de metástasis «escondidas» como de la quimiorresistencia. En el primer caso, hemos hipotetizado que los macrófagos «acompañan» en la circulación sanguínea a las células tumorales hasta los nichos metastásicos. En el segundo, tras observar que los macrófagos son los responsables de la eliminación de células apoptóticas, cuya producción aumenta exponencialmente tras la quimioterapia, hemos hipotetizado un mecanismo de retroalimentación negativa: tras la ingesta de células apoptóticas, los macrófagos secretan sustancias que reducen la sensibilidad a sucesivas apoptosis en el tejido tumoral. Para demostrar que los macrófagos se unen a células tumorales y las acompañan en la circulación sanguínea, hemos puesto a punto un sistema para detectar en biopsias líquidas y mediante citometría de flujo las macrocélulas formadas por macrófagos y las células tumorales. Así, hemos comparado el porcentaje de estas células en personas sanas y en otras con cáncer de páncreas, tanto en estadios precoces como en metastásicos. Los resultados demuestran que estas células solo se encuentran presentes en pacientes con cáncer metastásico. En el segundo caso, hemos incubado las células de cáncer de páncreas (PDAC) con medios condicionados de macrófagos que habían ingerido células apoptóticas (MØApop CM) y macrófagos sin el estímulo de células apoptóticas (MØCM). A continuación, se ha añadido un quimioterápico (gemcitabina o abraxane) a los dos medios condicionados, demostrando así que el MØApop CM confiere protección contra la quimioterapia. Se ha realizado posteriormente una proteómica que ha identificado la proteína YWHAZ/14-33 zeta/delta como potencial mediador del efecto del MØApop CM. Se han realizado experimentos utilizando la proteína 14-3-3ζ recombinante, que han demostrado que parte del efecto esta mediado esta. Asimismo, se ha demostrado que, in vivo, la eliminación de macrófagos de ratones con cáncer de páncreas mejora la respuesta al tratamiento quimioterápico. Por último, se ha demostrado que el mecanismo molecular por el cual la proteína 14-3-3ζ ejerce este efecto es mediante la integración con el receptor AX

A current perspective on cancer immune therapy: step-by-step approach to constructing the magic bullet

© The Author(s) 2017.Immunotherapy is the new trend in cancer treatment due to the selectivity, long lasting effects, and demonstrated improved overall survival and tolerance, when compared to patients treated with conventional chemotherapy. Despite these positive results, immunotherapy is still far from becoming the perfect magic bullet to fight cancer, largely due to the facts that immunotherapy is not effective in all patients nor in all cancer types. How and when will immunotherapy overcome these hurdles? In this review we take a step back to walk side by side with the pioneers of immunotherapy in order to understand what steps need to be taken today to make immunotherapy effective across all cancers. While early scientists, such as Coley, elicited an unselective but effective response against cancer, the search for selectivity pushed immunotherapy to the side in favor of drugs focused on targeting cancer cells. Fortunately, the modern era would revive the importance of the immune system in battling cancer by releasing the brakes or checkpoints (anti-CTLA-4 and anti-PD-1/PD-L1) that have been holding the immune system at bay. However, there are still many hurdles to overcome before immunotherapy becomes a universal cancer therapy. For example, we discuss how the redundant and complex nature of the immune system can impede tumor elimination by teeter tottering between different polarization states: one eliciting anti-cancer effects while the other promoting cancer growth and invasion. In addition, we highlight the incapacity of the immune system to choose between a fight or repair action with respect to tumor growth. Finally we combine these concepts to present a new way to think about the immune system and immune tolerance, by introducing two new metaphors, the “push the accelerator” and “repair the car” metaphors, to explain the current limitations associated with cancer immunotherapy.This work was supported by NIH R00 CA154605 and Louisiana Board of Regents LEQSF(2016-17)-RD-C-14 (H.L.M.), a Rámon y Cajal Merit Award from the Ministerio de Economía y Competitividad, Spain (B.S.Jr) and a Clinic and Laboratory Integration Program (CLIP) grant from the Cancer Research Institute, NY (B.S.Jr)

Age-Related Mortality in STEMI Patients: Insight from One Year of HUB Centre Experience during the Pandemic

Background: Old patients have a poor prognosis when affected by ST elevation myocardial infarction (STEMI). The aim of our study was to evaluate the impact of age on acute and mid-term mortality in STEMI patients over one year in the pandemic period. Methods: we collected data on 283 STEMI patients divided into three groups according to age (not old, &ldquo;Not-O&rdquo;, &le;74 y/o; old, &ldquo;O&rdquo;, 75&ndash;84 y/o; very old, &ldquo;Very-O&rdquo;, &ge;85 y/o). Results: the three groups did not differ in their clinical or procedural characteristics. The Very-O patients had a significantly increased incidence of in-hospital MACE (35%), mortality (30.0%), and percentage of cardiac death (25.0%). The only two independent predictors of in-hospital mortality were the ejection fraction (EF) [OR:0.902 (95% CI) 0.868&ndash;0.938; p &lt; 0.0001] and COVID-19 infection [OR:3.177 (95% CI) 1.212&ndash;8.331; p = 0.019]. At follow-up (430 +/&minus; days), the survival rates were decreased significatively among the age groups (Not-O 2.9% vs. O 14.8% vs. Very-O 28.6%; p &lt; 0.0001), and the only two independent predictors of the follow-up mortality were the EF [OR:0.935 (95% CI) 0.891&ndash;0.982; p = 0.007] and age [OR:1.06 (95% CI) 1.018&ndash;1.110; p = 0.019]. Conclusions: in very old patients, all the accessory procedures that may be performed should be accurately and independently weighed up in terms of the risk&ndash;benefit balance and the real impact on the quality of life because of the poor mid-term prognosis

Current evidence for cancer stem cells in gastrointestinal tumors and future research perspectives

Cancer stem cells (CSCs) are a very heterogeneous subpopulation of “stem-like” cancer cells that have been identified in many cancers, including leukemias and solid tumors. It is believed that CSCs drive tumor growth, malignant behavior and are responsible for the initiation of metastatic spread. In addition, CSCs have been implicated in chemotherapy and radiotherapy resistance. Current evidence supports the theory that CSCs share at least two main features of normal stem cells: self-renewal and differentiation, properties that contribute to tumor survival even in the presence of aggressive chemotherapy; however, the mechanism(s) governing the unique biology of CSCs remain unclear. In the field of gastrointestinal cancer, where we face very low survival rates across different tumor types, unraveling the role of CSCs in gastrointestinal tumors should improve our knowledge of cancer biology and chemoresistance, ultimately benefiting patient survival. Towards this end, much effort is being invested in the characterization of CSCs as a means of overcoming drug resistance and controlling metastatic spread. In this review we will cover the concept of CSCs, the current evidence for CSCs in gastrointestinal tumors and future research directions.This work was supported by a Ramon y Cajal Merit Award from the Ministerio de Economía y Competitividad, Spain (B.S. Jr) and a Clinic and Laboratory Integration Program (CLIP) grant from the Cancer Research Institute, NY (B.S. Jr)

Acoustic Voice Analysis as a Useful Tool to Discriminate Different ALS Phenotypes

Approximately 80–96% of people with amyotrophic lateral sclerosis (ALS) become unable to speak during the disease progression. Assessing upper and lower motor neuron impairment in bulbar regions of ALS patients remains challenging, particularly in distinguishing spastic and flaccid dysarthria. This study aimed to evaluate acoustic voice parameters as useful biomarkers to discriminate ALS clinical phenotypes. Triangular vowel space area (tVSA), alternating motion rates (AMRs), and sequential motion rates (SMRs) were analyzed in 36 ALS patients and 20 sex/age-matched healthy controls (HCs). tVSA, AMR, and SMR values significantly differed between ALS and HCs, and between ALS with prevalent upper (pUMN) and lower motor neuron (pLMN) impairment. tVSA showed higher accuracy in discriminating pUMN from pLMN patients. AMR and SMR were significantly lower in patients with bulbar onset than those with spinal onset, both with and without bulbar symptoms. Furthermore, these values were also lower in patients with spinal onset associated with bulbar symptoms than in those with spinal onset alone. Additionally, AMR and SMR values correlated with the degree of dysphagia. Acoustic voice analysis may be considered a useful prognostic tool to differentiate spastic and flaccid dysarthria and to assess the degree of bulbar involvement in ALS

Tumor-associated macrophage-secreted 14-3-3ζ signals via AXL to promote pancreatic cancer chemoresistance

Pancreatic ductal adenocarcinoma (PDAC) is an inherently chemoresistant tumor. Chemotherapy leads to apoptosis of cancer cells, and in previous studies we have shown that tumor-associated macrophage (TAM) infiltration increases following chemotherapy in PDAC. Since one of the main functions of macrophages is to eliminate apoptotic cells, we hypothesized that TAMs phagocytose chemotherapy-induced apoptotic cells and secrete factors, which favor PDAC chemoresistance. To test this hypothesis, primary human PDAC cultures were treated with conditioned media (CM) from monocyte-derived macrophage cultures incubated with apoptotic PDAC cells (MØApopCM). MØApopCM pretreatment rendered naïve PDAC cells resistant to Gemcitabine- or Abraxane-induced apoptosis. Proteomic analysis of MØApopCM identified YWHAZ/14-3-3 protein zeta/delta (14-3-3ζ), a major regulator of apoptotic cellular pathways, as a potential mediator of chemoresistance, which was subsequently validated in patient transcriptional datasets, serum samples from PDAC patients and using recombinant 14-3-3ζ and inhibitors thereof. Moreover, in mice bearing orthotopic PDAC tumors, the antitumor potential of Gemcitabine was significantly enhanced by elimination of TAMs using clodronate liposomes or by pharmacological inhibition of the Axl receptor tyrosine kinase, a 14-3-3ζ interacting partner. These data highlight a unique regulatory mechanism by which chemotherapy-induced apoptosis acts as a switch to initiate a protumor/antiapoptotic mechanism in PDAC via 14-3-3ζ/Axl signaling, leading to phosphorylation of Akt and activation of cellular prosurvival mechanisms. The data presented therefore challenge the idea that apoptosis of tumor cells is therapeutically beneficial, at least when immune sensor cells, such as macrophages, are presen