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Background: Next-generation sequencing (NGS) is recognised by a growing audience of medical professionals as a functional diagnostic tool in oncology. However, adoption in clinical routine proceeds haphazardly in Europe., Methods: A semi-structured interview survey was administered to 68 cancer care professionals in four EU countries between June-August and November-December 2021. Pre-screening questionnaires assessed sufficient NGS expertise, diverse geographical distribution, and professional roles., Results: Our findings provide a better understanding of current clinical, regulatory, and reimbursement practices for NGS in four EU countries., Conclusions: Despite the impending European In-vitro Diagnostic Medical Devices Regulation (IVDR), tortuous national guidelines implementations and limping reimbursement policies are common traits across surveyed countries and produce disparity in access to advanced healthcare services amid regional distinctions., Policy Summary: The evident information gap between involved parties and demand for consistent national guidelines could be filled by health economics analyses tailored to local specifics to provide factual leverage for a structured adoption of NGS testing., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Brazil is the third largest contributor to Green Analytical Chemistry, and there is significant participation of toxicologists in the development and improvement of environmental techniques. Currently, toxicologists have their own strategies and guidelines to promote the reduction/replacement or elimination of solvents, reduce the impacts of derivatization and save time, among other objectives, due to the peculiarities of toxicological analysis. Thus, this review aims to propose the concept of Green Analytical Toxicology and conduct a discussion about its relevance and applications specifically in forensic toxicology, using the microextraction methods developed for the determination of cocaine and its metabolites as examples., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Purpose: MDA-19 or BZO-HEXOXIZID (N'-[(3Z)-1-(1-hexyl)-2-oxo-1,2-dihydro-3H-indol-3-ylidene]-benzohydrazide), in a more recent nomenclature, was first synthesized in 2008 as a selective type-2 cannabinoid receptor (CB2) agonist due to its potential to treat neuropathic pain. In Brazil, this substance was identified in a series of 53 apprehensions between September 2021 and February 2022. Nevertheless, what intrigues toxicologists is that BZO-HEXOXIZID does not exert significant type-1 cannabinoid receptor (CB1) agonism-which is responsible for the well-known psychoactivity of Δ-9-tetrahydrocannabinol. Thus, the objective of this work is to report the first apprehension and identification of BZO-HEXOXIZID in Brazil and to discuss pharmacologically the possible reasons why a CB2 agonist has been incorporated to the illicit market., Methods: Suspected seized samples were sent to the Laboratory of the Scientific Police of the State of Sao Paulo. After the screening, samples were confirmed for the presence of BZO-HEXOXIZID using chromatography gas-mass spectrometry, Fourier-transform infrared spectroscopy and nuclear magnetic resonance techniques., Results: Of the 53 samples analyzed, 25 contained only BZO-HEXOXIZID and 28 with mixtures, of which 11 with the CB1 agonist ADB-BUTINACA. Other substances were found in association such as cocaine and caffeine., Conclusions: BZO-HEXOXIZID was detected in a series of seized materials for the first time in Brazil. Nevertheless, there are still unanswered questions regarding the use of this selective CB2 agonist as a drug of abuse., (© 2022. The Author(s), under exclusive licence to Japanese Association of Forensic Toxicology.)

Lucy in the Sky with Diamonds, a John Lennon song that was a hit in the 1960s, was born amidst a social context enlightened by lysergic acid diethylamide (LSD). In Brazil, both the drug and the song were very popular at the time, although it gradually mitigated. Nevertheless, while the song remains out of the spotlight, LSD derivatives are currently gaining attention with the rising of the new psychoactive substances (NPS). With this new presentation, the drug is returning to Brazil after a few decades and herein we report and discuss the first cases of an LSD prodrug seized in our country. Nine suspected blotter paper samples were seized by the Sao Paulo State Police in different cities of the State. Gas chromatography-mass spectrometry (GC-MS), attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) and liquid chromatography coupled with electrospray ionization-mass spectrometry (LC-ESI-MS) analyses were utilized to confirm the identity of the LSD derivative. The compound was identified as 4-acetyl-N,N-diethyl-7-methyl-4,6,6a,7,8,9-hexahydroindolo[4,3-fg]quinoline-9-carboxamide (ALD-52 or 1A-LSD) and no other active substance was detected in all samples. The identity of the unknown compound found in seized blotter papers has been successfully confirmed as an LSD prodrug, ALD-52, which was not controlled by Brazilian legislation. The arrival of a new type of designer drug in Brazil is in support by other reports, although those are still scarce and should not be overlooked. Altogether, these findings indicate the rising of a new NPS strategy that merits proper discussion., Competing Interests: Conflict of interest The authors have declared no conflict of interest., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Cannabis is the most widely used illegal drug during pregnancy, however, the effects of gestational exposure to Cannabis smoke (CS) on the central nervous system development remain uncharacterised. This study investigates the effects of maternal CS inhalation on brain function in the offspring. Pregnant mice were exposed daily to 5 min of CS during gestational days (GD) 5.5-17.5. On GD 18.5 half of the dams were euthanized for foetus removal. The offspring from the remaining dams were euthanized on postnatal days (PND) 20 and 60 for evaluation. Brain volume, cortex cell number, SOX2, histone-H3, parvalbumin, NeuN, and BDNF immunoreactivity were assessed in all groups. In addition, levels of NeuN, CB1 receptor, and BDNF expression were assessed and cortical primary neurons from rats were treated with Cannabis smoke extract (CSE) for assessment of cell viability. We found that male foetuses from the CS exposed group had decreased brain volume, whereas mice at PND 60 from the exposed group presented with increased brain volume. Olfactory bulb and diencephalon volume were found lower in foetuses exposed to CS. Mice at PND 60 from the exposed group had a smaller volume in the thalamus and hypothalamus while the cerebellum presented with a greater volume. Also, there was an increase in cortical BDNF immunoreactivity in CS exposed mice at PND 60. Protein expression analysis showed an increase in pro-BDNF in foetus brains exposed to CS. Mice at PND 60 presented an increase in mature BDNF in the prefrontal cortex (PFC) in the exposed group and a higher CB1 receptor expression in the PFC. Moreover, hippocampal NeuN expression was higher in adult animals from the exposed group. Lastly, treatment of cortical primary neurons with doses of CSE resulted in decreased cell viability. These findings highlight the potential negative neurodevelopmental outcomes induced by gestational CS exposure., Competing Interests: Declarations of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The aim of this study was to develop a quantitative method for the analysis of methylphenidate, the analog ethylphenidate and their metabolite ritalinic acid in oral fluid, using micro-QuEChERS extraction and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Oral fluid samples were collected with Quantisal™ device, extracted by micro-QuEChERS technique and analyzed by LC-MS/MS. The developed method met the validation criteria of Academy Standards Board (ASB) Standard Practices for Method Validation in Forensic Toxicology (Standard 036, 2019) with limits of detection and quantification of 0.5 ng/mL and calibration curve from 0.5 to 50 ng/mL. Within-run imprecision was greater than 18.7% while between-run imprecision was greater than 17.0 % for all analytes. Bias did not vary more than 7.7 %. No evidence of carryover was found. Stability studies presented satisfactory results for 24 h on autosampler (10 °C), after 3 cycles of freeze/thaw, 7 days on freezer (-20 °C) and until 7 days on refrigerator (4 °C) for methylphenidate. The validated method was further successfully applied to the analysis of 5 authentic oral fluid samples collected from volunteers at parties and music festivals from different cities in Brazil. Four samples had positive results for methylphenidate and ritalinic acid, and only one sample was positive for methylphenidate. Ethylphenidate was not detected in the samples. The method showed acceptable analytical performance and is environmentally friendly, requiring reduced use of solvents and reagents, with potential to be applied to clinical and forensic analyses., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Anthropogenic pressures such as the introduction of non-indigenous species (NIS) have impacted global biodiversity and ecosystems. Most marine species spreading outside their natural biogeographical limits are promoted and facilitated by maritime traffic through ballast water and hull biofouling. Propagule pressure plays a primary role in invasion success mixed with environmental conditions of the arrival port. Moreover, with the current ocean sprawl, new substrates are offered for potential NIS recruits. Here, differences in the fouling assemblages thriving inside three different ports/marinas facilities in Madeira Island were assessed for comparison. The locations showed significant differences concerning assemblage structure. Most NIS were detected in plastic floating pontoons. Funchal harbour receives most of the marine traffic in Madeira, acting as the main hub for primary NIS introductions, being recreational boating involved in NIS secondary transfers. Our results highlight the need for future management actions in island ecosystems, particularly monitoring and sampling of recreational boating., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Cannabinoids are pharmacologically active compounds present in cannabis plants, which have become important research topics in the modern toxicological and medical research fields. Not only is cannabis the most used drug globally, but also cannabinoids have a growing use to treat a series of diseases. Therefore, new, fast, and efficient analytical methods for analyzing these substances in different matrices are demanded. This study developed a new packed-in-tube solid-phase microextraction (IT-SPME) method coupled to liquid chromatography with tandem mass spectrometry (LC-MS/MS), for the automated microextraction of seven cannabinoids from human urine. Packed IT-SPME microcolumns were prepared in (508 µm i.d. × 50 mm) stainless-steel hardware; each one required only 12 mg of sorbent phase. Different sorbents were evaluated; fractional factorial design 2 4-1 and a central composite design were employed for microextraction optimization. Under optimized conditions, the developed method was a fast and straightforward approach. Only 250 µl of urine sample was needed, and no hydrolysis was required. The sample pretreatment included only dilution and centrifugation steps (8 min), whereas the complete IT-SPME-LC-MS/MS method took another 12 min, with a sample throughput of 3 samples h -1 . The developed method presented adequate precision, accuracy and linearity; R 2 values ranged from 0.990 to 0.997, in the range of 10-1000 ng ml -1 . The lower limits of quantification varied from 10 to 25 ng ml -1 . Finally, the method was successfully applied to analyze 20 actual urine samples, and the IT-SPME microcolumn was reused over 150 times., (© 2022 Wiley-VCH GmbH.)

Purpose: We have developed and validated a high-sensitivity method to quantify lysergic acid diethylamide (LSD) and 2-oxo-3-hydroxy-LSD (OH-LSD) in oral fluid samples using liquid-liquid extraction and liquid chromatography-tandem mass spectrometry (LC‒MS/MS). The method was applied to the quantification of both substances in 42 authentic oral fluid samples., Methods: A liquid-liquid extraction was performed using 500 µL each of samples (oral fluid samples collected using Quantisal™ device) and dichloromethane/isopropanol mixture (1:1, v/v). Enzymatic hydrolysis was evaluated to cleave glucuronide metabolites., Results: The limit of quantification was 0.01 ng/mL for both LSD and OH-LSD. The linearity was assessed between 0.01 and 5 ng/mL. Imprecision and bias were not higher than 10.2% for both analytes. Extraction recovery was higher than 69%. The analytes were stable in the autosampler at 10 °C for 24 h, and up to 30 days at 4 and -20 °C. The method was applied to the analysis of 42 oral fluid samples. LSD was detected in all samples (concentrations between 0.02 and 175 ng/mL), and OH-LSD was detected in 20 samples (concentrations between 0.01 and 1.53 ng/mL)., Conclusions: A high-sensitive method was fully validated and applied to authentic samples. To our knowledge, this is the first work to report concentrations of LSD and OH-LSD in authentic oral fluid samples., (© 2022. The Author(s), under exclusive licence to Japanese Association of Forensic Toxicology.)

Background: Coronavirus disease 2019 (COVID-19) is caused by a novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It is known that host microRNAs (miRNAs) can be modulated to favor viral infection or to protect the host. Herein, we report preliminary results of a study aiming at identifying differentially expressed plasmatic miRNAs in Brazilian patients with COVID-19., Methods and Results: miRNAs were extracted from the plasma of eight patients with COVID-19 (four patients with mild COVID-19 and four patients with severe/critical COVID-19) and four healthy controls. Patients and controls were matched for sex and age. miRNA expression levels were detected using high-throughput sequencing. Differential miRNA expression and enrichment analyses were further evaluated. A total of 18 miRNAs were differentially expressed between patients with COVID-19 and controls. miR-4433b-5p, miR-6780b-3p, miR-6883-3p, miR-320b, miR-7111-3p, miR-4755-3p, miR-320c, and miR-6511a-3p were the most important miRNAs significantly involved in the PI3K/AKT, Wnt/β-catenin, and STAT3 signaling pathways. Moreover, 42 miRNAs were differentially expressed between severe/critical and mild patients with COVID-19. miR-451a, miR-101-3p, miR-185-5p, miR-30d-5p, miR-25-3p, miR-342-3p, miR-30e-5p, miR-150-5p, miR-15b-5p, and miR-29c-3p were the most important miRNAs significantly involved in the Wnt/β-catenin, NF-κβ, and STAT3 signaling pathways., Conclusions: If validated by quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR) in a larger number of participants, the miRNAs identified in this study might be used as possible biomarkers for the diagnosis and severity of COVID-19., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

Background: Thrombotic antiphospholipid syndrome (t-PAPS) is characterized by arterial, venous, or microvascular occlusions, which are explained, in part, by the presence of antiphospholipid (aPL) antibodies. Although there is much evidence indicating that isolated aPL antibodies increase the activity of platelets obtained from healthy volunteers, platelet function in t-PAPS has not been as widely studied., Objective: To evaluate platelet reactivity in t-PAPS patients., Methods: Platelet aggregation, protein expression, and cyclic nucleotide levels were carried out in platelet rich plasma (PRP) or washed platelets (WPs) obtained from t-PAPS or healthy volunteers., Results: ADP-induced aggregation was significantly higher in PRP obtained from t-PAPS than obtained from the control. The protein expression of P2Y 12 receptor and Gs alpha was significantly higher and lower, respectively in WPs from t-PAPS patients. In PRP incubated with iloprost or sodium nitroprusside, the residual platelet reactivity induced by ADP was still higher in PRP from t-PAPS than from the control. Lower intracellular levels of cyclic guanosine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP) were observed in unstimulated PRP from t-PAPS patients. The protein expression of soluble guanylate cyclase subunits and phosphodiesterases types 3 and 5 did not differ. The antiplatelet activity of ticagrelor was similar between the groups and cilostazol significantly potentiated this response. Isolated aPL antibodies obtained from t-PAPS patients potentiated ADP-induced aggregation in healthy platelets but did not affect the inhibitory responses induced by iloprost or sodium nitroprusside., Conclusions: The overexpression of P2Y 12 receptor, accompanied by lower levels of cAMP and cGMP levels produced greater amplitude of ADP aggregation in platelets from t-PAPS patients., (© 2022 International Society on Thrombosis and Haemostasis.)

Witches' broom disease of cacao is caused by the pathogenic fungus Moniliophthora perniciosa. By using tomato (Solanum lycopersicum) cultivar Micro-Tom (MT) as a model system, we investigated the physiological and metabolic consequences of M. perniciosa infection to determine whether symptoms result from sink establishment during infection. Infection of MT by M. perniciosa caused reductions in root biomass and fruit yield, a decrease in leaf gas exchange, and down-regulation of photosynthesis-related genes. The total leaf area and water potential decreased, while ABA levels, water conductance/conductivity, and ABA-related gene expression increased. Genes related to sugar metabolism and those involved in secondary cell wall deposition were up-regulated upon infection, and the concentrations of sugars, fumarate, and amino acids increased. 14C-glucose was mobilized towards infected MT stems, but not in inoculated stems of the MT line overexpressing CYTOKININ OXIDASE-2 (35S::AtCKX2), suggesting a role for cytokinin in establishing a sugar sink. The up-regulation of genes involved in cell wall deposition and phenylpropanoid metabolism in infected MT, but not in 35S::AtCKX2 plants, suggests establishment of a cytokinin-mediated sink that promotes tissue overgrowth with an increase in lignin. Possibly, M. perniciosa could benefit from the accumulation of secondary cell walls during its saprotrophic phase of infection., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Lung diseases affect the lives of billions of people worldwide, and 4 million people, each year, die prematurely due to this condition. These pathologies are characterized by specific imagiological findings in CT scans. The traditional Computer-Aided Diagnosis (CAD) approaches have been showing promising results to help clinicians; however, CADs normally consider a small part of the medical image for analysis, excluding possible relevant information for clinical evaluation. Multiple Instance Learning (MIL) approach takes into consideration different small pieces that are relevant for the final classification and creates a comprehensive analysis of pathophysiological changes. This study uses MIL-based approaches to identify the presence of lung pathophysiological findings in CT scans for the characterization of lung disease development. This work was focus on the detection of the following: Fibrosis, Emphysema, Satellite Nodules in Primary Lesion Lobe, Nodules in Contralateral Lung and Ground Glass, being Fibrosis and Emphysema the ones with more outstanding results, reaching an Area Under the Curve (AUC) of 0.89 and 0.72, respectively. Additionally, the MIL-based approach was used for EGFR mutation status prediction - the most relevant oncogene on lung cancer, with an AUC of 0.69. The results showed that this comprehensive approach can be a useful tool for lung pathophysiological characterization., (© 2022. International Federation for Medical and Biological Engineering.)

Bearing in mind the several medicinal properties of Mentha genus, this work aimed to evaluate the anti-proliferative potential of the ethanolic extract (EE) and fractions from M. aquatica L aerial parts. Using the anti-proliferative protocol developed by the NCI/USA, four fractions (F2 - F4 and F6) obtained from EE showed promising anti-proliferative profile against a panel of human tumor and non-tumor cell lines. After 24-h exposure, F2 (0.25 µg/mL) showed potent and irreversible anti-proliferative effect without inducing cell cycle arrest in both NCI-H460 and MCF-7 cells, without (anti) estrogenic activity. These effects were lost after storage of F2 diluted in dimethyl sulfoxide at -80 °C during 2 weeks. Analysis by gas chromatography coupled to mass detection evidenced some chemical changes induced by F2 storage in solution. The present study demonstrated the anti-proliferative effect of M. aquatica . Further studies are necessary to determine better storage conditions to enhance F2 stability.

Considering that the use of psychoactive substances (PSs) is a risk factor to either higher intensity or frequency of suicidal behavior, hair analysis was conducted to investigate the most consumed PSs (opiates, amphetamine stimulants, marijuana, cocaine and heroin) in patients who attempted suicide and received urgent care at emergency service. Hair samples were extracted using methanol and sonicated under heating and then analyzed using liquid chromatography-tandem mass spectrometry. During validation, the method complied with international recommended criteria, with limits of detection between 0.0025 and 0.05 ng/mg and linearity between 0.1 and 4 ng/mg for methamphetamine, 3,4-methylenedioxymethamphetamine (MDMA), morphine, amphetamine, 6-acetylmorphine, 3,4-methylenedioxyamphetamine (MDA), fenproporex, diethylpropion and codeine; between 0.025 and 1 ng/mg for tetrahydrocannabinol (THC), benzoylecgonine and cocaethylene and between 0.25 and 10 ng/mg for cocaine and mazindol. A total of 109 hair samples were analyzed and segmented in 404 parts. Among all analyzed samples, 30.3% were positive for at least one PS (n = 33), such as cocaine (90.9%), codeine (12.1%), morphine (3.0%), MDMA (3.0%) and THC (3.0%). In segmental analysis of cocaine positive samples (n = 30), 76.7% of the samples indicated recent exposure to cocaine (<1 month). This same behavior was observed when analyzing codeine (n = 4) and morphine (n = 1). THC positive samples indicated exposure dated ∼4 months prior. In conclusion, the method was validated following international recommendations for the 12 most consumed PSs in Brazil, as well as two of the most common found metabolites., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Tomato production is influenced by shoot branching, which is controlled by different hormones. Here we produced tomato plants overexpressing the cytokinin-deactivating gene CYTOKININ OXYDASE 2 (CKX2). CKX2-overexpressing (CKX2-OE) plants showed an excessive growth of axillary shoots, the opposite phenotype expected for plants with reduced cytokinin content, as evidenced by LC-MS analysis and ARR5-GUS staining. The TCP transcription factor SlBRC1b was downregulated in the axillary buds of CKX2-OE and its excessive branching was dependent on a functional version of the GRAS-family gene LATERAL SUPPRESSOR (LS). Grafting experiments indicated that increased branching in CKX2-OE plants is unlikely to be mediated by root-derived signals. Crossing CKX2-OE plants with transgenic antisense plants for the strigolactone biosynthesis gene CAROTENOID CLEAVAGE DIOXYGENASE (CCD7-AS) produced an additive phenotype, indicating independent effects of cytokinin and strigolactones on increased branching. On the other hand, CKX2-OE plants showed reduced polar auxin transport and their bud outgrowth was reduced when combined with auxin mutants. Accordingly, CKX2-OE basal buds did not respond to auxin applied in the decapitated apex. Our results suggest that tomato shoot branching depends on a fine-tuning of different hormonal balances and that perturbations in the auxin status could compensate for the reduced cytokinin levels in CKX2-OE plants., (© 2022. The Author(s).)

Advancements in the development of computer-aided decision (CAD) systems for clinical routines provide unquestionable benefits in connecting human medical expertise with machine intelligence, to achieve better quality healthcare. Considering the large number of incidences and mortality numbers associated with lung cancer, there is a need for the most accurate clinical procedures; thus, the possibility of using artificial intelligence (AI) tools for decision support is becoming a closer reality. At any stage of the lung cancer clinical pathway, specific obstacles are identified and "motivate" the application of innovative AI solutions. This work provides a comprehensive review of the most recent research dedicated toward the development of CAD tools using computed tomography images for lung cancer-related tasks. We discuss the major challenges and provide critical perspectives on future directions. Although we focus on lung cancer in this review, we also provide a more clear definition of the path used to integrate AI in healthcare, emphasizing fundamental research points that are crucial for overcoming current barriers.

Objective: Intratumor heterogeneity drives cancer progression and therapy resistance. However, it has yet to be determined whether and how subpopulations of cancer cells interact and how this interaction affects the tumour., Design: We have studied the spontaneous flow of extracellular vesicles (EVs) between subpopulations of cancer cells: cancer stem cells (CSC) and non-stem cancer cells (NSCC). To determine the biological significance of the most frequent communication route, we used pancreatic ductal adenocarcinoma (PDAC) orthotopic models, patient-derived xenografts (PDXs) and genetically engineered mouse models (GEMMs)., Results: We demonstrate that PDAC tumours establish an organised communication network between subpopulations of cancer cells using EVs called the EVNet). The EVNet is plastic and reshapes in response to its environment. Communication within the EVNet occurs preferentially from CSC to NSCC. Inhibition of this communication route by impairing Rab27a function in orthotopic xenographs, GEMMs and PDXs is sufficient to hamper tumour growth and phenocopies the inhibition of communication in the whole tumour. Mechanistically, we provide evidence that CSC EVs use agrin protein to promote Yes1 associated transcriptional regulator (YAP) activation via LDL receptor related protein 4 (LRP-4). Ex vivo treatment of PDXs with antiagrin significantly impairs proliferation and decreases the levels of activated YAP.Patients with high levels of agrin and low inactive YAP show worse disease-free survival. In addition, patients with a higher number of circulating agrin + EVs show a significant increased risk of disease progression., Conclusion: PDAC tumours establish a cooperation network mediated by EVs that is led by CSC and agrin, which allows tumours to adapt and thrive. Targeting agrin could make targeted therapy possible for patients with PDAC and has a significant impact on CSC that feeds the tumour and is at the centre of therapy resistance., Competing Interests: Competing interests: SAM holds patents in the area of exosome biology. TK is a founder of Abcam plc and Storm Therapeutics and is on the scientific advisory board of Foghorn Therapeutics. RK holds patents in the area of exosomes biology and are licensed to Codiak Biosciences, Inc. RK is stockholder in Codiak Biosciences, Inc. RK is a consultant and scientific adviser for Codiak Biosciences, Inc. The other authors declare no potential conflict of interest., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Aim: THC-COOH is the major metabolite of Δ 9 -tetrahydrocannabinol commonly tested in urine to determine cannabis intake. In this study, a method based on dispersive liquid-liquid microextraction was developed for testing THC-COOH in urine. Materials & methods: Hydrolyzed urine specimens were extracted via dispersive liquid-liquid microextraction with acetonitrile (disperser solvent) and chloroform (extraction solvent). Derivatization was performed with N,O -Bis(trimethylsilyl)trifluoroacetamide with 1% trichloro(chloromethyl)silane. Analysis was performed by GC-MS/MS. Results: The method showed acceptable linearity (5-500 ng/ml), imprecision (<10.5%) and bias (<4.9%). Limits of detection and quantitation were 1 and 5 ng/ml, respectively. Twenty-four authentic samples were analyzed, with 22 samples being positive for THC-COOH. Conclusion: The proposed method is more environmentally friendly and provided good sensitivity, selectivity and reproducibility.

Retention is one of the processes controlling the behavior and fate of pesticides in soil. The objective of this work was to evaluate the adsorption and desorption of glyphosate, AMPA, and metsulfuron-methyl in the main horizons of a Typic Argiudoll destined for agricultural use. For this purpose, the batch equilibrium method was used at a range of concentrations for each compound. Desorption was performed in three consecutive steps after the adsorption experiment. The results obtained showed strong adsorption of glyphosate and AMPA in the three horizons, following the trend B > A > C, with weak desorption. Metsulfuron-methyl, on the other hand, showed weak adsorption in the three horizons, following the trend A > B > C, with relevant desorption. Our results allow us to identify metsulfuron-methyl as the compound that poses the greatest environmental risk in terms of the potential contamination of other areas and groundwater. However, despite their strong adsorption and weak desorption, glyphosate and AMPA also represent potential contaminants of other environmental matrices.

The human chorionic gonadotropin (hCG) stimulation test that evaluates gonadal steroidogenesis is crucial in the assessment of patients with 46,XY disorders of sex development (DSD). This study aimed to determine a testosterone (T) cutoff level that indicates an adequate testicular function using LC-MS/MS after stimulation with recombinant human chorionic gonadotropin (rhCG) in a single dose. Nineteen prepubertal children with 46,XY DSD and normal T secretion were evaluated. T and dihydrotestosterone (DHT) levels were measured by liquid chromatography technique with tandem mass spectrometry (LC-MS/MS) before and 7 days after rhCG application at 250 µg. We suggest 0.89 ng/mL as the cutoff point for T after rhCG stimulation analyzed by LC-MS/MS., (© 2021 S. Karger AG, Basel.)

Purpose: Synthetic cannabinoid receptor agonists (SCRAs) are a class of varied compounds that mimic the effects of natural cannabinoids found in cannabis. Because they have a wide range of diverse structures, they vary widely in their potency. The abuse of new psychoactive substances (NPS) in prisons was reported in many European countries and in the USA. In the present study, we have described the identification of SCRAs in 56 infused paper sheet samples, seized mainly in Brazilian prisons between 2016 and 2020., Methods: The materials were seized by local or federal law enforcement and analyzed by São Paulo State Police or Brazilian Federal Police using gas chromatography-mass spectrometry, attenuated total reflection-Fourier transform infrared spectroscopy, liquid chromatography-high-resolution mass spectrometry or nuclear magnetic resonance spectrometry., Results: Most of these samples (87.5%) were seized in 2019-2020; seven different SCRAs were identified in samples, and the most frequently identified substances were MDMB-4en-PINACA (23.6%) and 5F-MDMB-PICA (36.4%), the newest SCRAs emerging recently., Conclusions: As observed in Europe and the USA, Brazil also shows the prevalence of indazole-3-carboxamides and indole-3-carboxamides among SCRAs seizures in the prison system. This phenomenon is spreading all over the world at this moment. These data on the prevalence could help to alert judicial authorities to shutting down the introduction of NPS, including SCRAs, into prisons to ensure safety and security for avoiding health risks of prisoners and staff, leading to positive effects in this population. To our knowledge, this is the first demonstration of SCRAs smuggling into prisons in Latin America., (© 2021. Japanese Association of Forensic Toxicology.)

Liquid biopsy (LB) has boosted a remarkable change in the management of cancer patients by contributing to tumour genomic profiling. Plasma circulating cell-free tumour DNA (ctDNA) is the most widely searched tumour-related element for clinical application. Specifically, for patients with lung cancer, LB has revealed valuable to detect the diversity of targetable genomic alterations and to detect and monitor the emergence of resistance mechanisms. Furthermore, its non-invasive nature helps to overcome the difficulty in obtaining tissue samples, offering a comprehensive view about tumour diversity. However, the use of the LB to support diagnostic and therapeutic decisions still needs further clarification. In this sense, this review aims to provide a critical view of the clinical importance of plasma ctDNA analysis, the most widely applied LB, and its limitations while anticipating concepts that will intersect the present and future of LB in non-small cell lung cancer patients., (© 2021. The Author(s).)

The production of cupped oysters is an important component of European aquaculture. Most of the production relies on the cultivation of the Pacific oyster Crassostrea gigas , although the Portuguese oyster Crassostrea angulata represents a valuable product with both cultural and economic relevance, especially in Portugal. The authors of the present study investigated the genetic diversity of Portuguese oyster populations of the Sado estuary, both from natural oyster beds and aquaculture facilities, through cox1 gene fragment sequencing. Then, a comparison with a wide dataset of cupped oyster sequences obtained from GenBank (up to now the widest available dataset in literature for the Portuguese oyster) was performed. Genetic data obtained from this work confirmed that the Pacific oyster does not occur in the natural oyster beds of the Sado estuary but showed that the species occasionally occurs in the oyster hatcheries. Moreover, the results showed that despite the founder effect and the bottleneck events that the Sado populations have experienced, they still exhibit high haplotype diversity. Risks are arising for the conservation of the Portuguese oyster reference populations of the Sado estuary due to the occurrence of the Pacific oyster in the local hatcheries. Therefore, researchers, local authorities, and oyster producers should work together to avoid the loss of this valuable resource.

Lung cancer is the deadliest form of cancer, accounting for 20% of total cancer deaths. It represents a group of histologically and molecularly heterogeneous diseases even within the same histological subtype. Moreover, accurate histological subtype diagnosis influences the specific subtype's target genes, which will help define the treatment plan to target those genes in therapy. Deep learning (DL) models seem to set the benchmarks for the tasks of cancer prediction and subtype classification when using gene expression data; however, these methods do not provide interpretability, which is great concern from the perspective of cancer biology since the identification of the cancer driver genes in an individual provides essential information for treatment and prognosis. In this work, we identify some limitations of previous work that showed efforts to build algorithms to extract feature weights from DL models, and we propose using tree-based learning algorithms that address these limitations. Preliminary results show that our methods outperform those of related research while providing model interpretability.Clinical Relevance: The machine learning methods used in this work are interpretable and provide biological insight. Two sets of genes were extracted: a set that differentiates normal tissue from cancerous tissue (cancer prediction), and a set of genes that distinguishes LUAD from LUSC samples (subtype classification).

Deep Neural Networks using histopathological images as an input currently embody one of the gold standards in automated lung cancer diagnostic solutions, with Deep Convolutional Neural Networks achieving the state of the art values for tissue type classification. One of the main reasons for such results is the increasing availability of voluminous amounts of data, acquired through the efforts employed by extensive projects like The Cancer Genome Atlas. Nonetheless, whole slide images remain weakly annotated, as most common pathologist annotations refer to the entirety of the image and not to individual regions of interest in the patient's tissue sample. Recent works have demonstrated Multiple Instance Learning as a successful approach in classification tasks entangled with this lack of annotation, by representing images as a bag of instances where a single label is available for the whole bag. Thus, we propose a bag/embedding-level lung tissue type classifier using Multiple Instance Learning, where the automated inspection of lung biopsy whole slide images determines the presence of cancer in a given patient. Furthermore, we use a post-model interpretability algorithm to validate our model's predictions and highlight the regions of interest for such predictions.

Background: New psychoactive substances (NPS) use is a worldwide public health issue. Knowing the prevalence of NPS guides public health and legal policies to address the problem. The objective of this study was to identify NPS in Brazil through the analysis of oral fluid (OF) samples collected at parties and electronic music festivals., Methods: Anonymous questionnaires and oral fluid samples were collected from volunteers (≥18 years) who reported the consumption of at least one illicit psychoactive substance in the last 24 h. Oral fluid sample collections occurred at eleven parties and two electronic music festivals over 16 months (2018-2020). Questionnaire answers were matched to oral fluid toxicological results., Results: Of 462 oral fluid samples, 39.2 % were positive for at least one NPS by liquid chromatography‒tandem mass spectrometry (LC-MS/MS). The most prevalent NPS was ketamine (29.4 %), followed by methylone (6.1 %) and N-ethylpentylone (4.1 %); however, MDMA was the most commonly identified (88.5 %) illicit psychoactive substance. More than one drug was identified in 79.9 % of samples, with two (34.2 %) and three (23.4 %) substances most commonly observed. Only 5 % of volunteers reported recent NPS consumption., Conclusion: MDMA is still the most common party and electronic music festival drug, although NPS were identified in more than one-third of oral fluid samples., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background: The tumor immune microenvironment exerts a pivotal influence in tumor initiation and progression. The aim of this study was to analyze the immune context of sporadic and familial adenomatous polyposis (FAP) lesions along the colorectal adenoma-carcinoma sequence (ACS)., Methods: We analyzed immune cell counts (CD3+, CD4+, CD8+, Foxp3+, and CD57+), tumor mutation burden (TMB), MHC-I expression and PD-L1 expression of 59 FAP and 74 sporadic colorectal lesions, encompassing adenomas with low-grade dysplasia (LGD) (30 FAP; 30 sporadic), adenomas with high-grade dysplasia (22 FAP; 30 sporadic), and invasive adenocarcinomas (7 FAP; 14 sporadic)., Results: The sporadic colorectal ACS was characterized by (1) a stepwise decrease in immune cell counts, (2) an increase in TMB and MHC-I expression, and (3) a lower PD-L1 expression. In FAP lesions, we observed the same patterns, except for an increase in TMB along the ACS. FAP LGD lesions harbored lower Foxp3+ T cell counts than sporadic LGD lesions. A decrease in PD-L1 expression occurred earlier in FAP lesions compared to sporadic ones., Conclusions: The colorectal ACS is characterized by a progressive loss of adaptive immune infiltrate and by the establishment of a progressively immune cold microenvironment. These changes do not appear to be related with the loss of immunogenicity of tumor cells, or to the onset of an immunosuppressive tumor microenvironment.

Background and Purpose: Advances in the treatment of triple-negative breast and ovarian cancer remain challenging. In particular, resistance to the available therapy, by restoring or overexpressing the DNA repair machinery, has often been reported. New strategies to improve the therapeutic outcomes of these cancers are needed. Herein, we disclose the dregamine 5-bromo-pyridin-2-ylhydrazone (BBIT20), a natural monoterpene indole alkaloid derivative, as an inhibitor of homologous DNA repair., Experimental Approach: To unveil BBIT20 antitumour activity and underlying molecular mechanism of action, two-dimensional (2D) and three-dimensional (3D) cell cultures, patient-derived cell lines and xenograft mouse models were used., Key Results: BBIT20 disrupted the BRCA1-BARD1 interaction, triggering nuclear-to-cytoplasmic BRCA1 translocation, cell cycle arrest and downregulation of homologous DNA repair-related genes and proteins, with subsequent enhancement of DNA damage, reactive oxygen species generation and apoptosis, in triple-negative breast and ovarian cancer cells. BBIT20 also displayed pronounced antitumour activity in patient-derived cells and xenograft mouse models of ovarian cancer, with low toxicity in non-malignant cells and undetectable side effects in mice. Additionally, it did not induce resistance in triple-negative breast and ovarian cancer and displayed marked synergistic effects with cisplatin and olaparib (a poly [ADP-ribose] polymerase inhibitor), on 2D and 3D models of these cancer cells., Conclusion and Implications: These findings add an inhibitor of the BRCA1-BARD1 interaction to the list of DNA-damaging agents. Importantly, either as a single agent or in combination therapy, BBIT20 reveals great potential in the personalized treatment of aggressive and resistant cancers, particularly triple-negative breast and advanced ovarian cancer., (© 2021 The British Pharmacological Society.)

The development of new sample preparation alternatives in analytical toxicology leading to quick, effective, automated and environmentally friendly procedures is growing in importance. One of these alternatives is the QuEChERS, originally developed for the analysis of pesticide residues, producing cleaner extracts than liquid-liquid extraction, and easier separation of aqueous and organic phases. However, there are few published studies on the miniaturization of this technique for forensic toxicology, especially in postmortem analysis. We developed and validated a modified micro-QuEChERS and LC-MS-MS assay to quantify 16 antidepressants, 7 antipsychotics and 3 metabolites and semi-quantify norfluoxetine and norsertraline in postmortem blood. The calibration curve was linear from 1 to 500 ng/mL, achieved an r > 0.99, with all standards quantifying within ±15% of target except ±20% at the limit of quantification of 1 ng/mL for 26 substances. The F test was applied to evaluate if the variance between replicates remained constant for all calibrators. Six weighting factors were analyzed (1/x, 1/x2, 1/x0,5, 1/y, 1/y2 and 1/y0,5), with the weighting factor with the lowest sum of residual regression errors (1/x2) selected. No endogenous or exogenous interferences were observed. Method imprecision and bias were <19.0% and 19.7%, respectively. Advantages of this method include a low sample volume of 100 µL, simple but effective sample preparation and a rapid 8.5-min run time. The validated analytical method was successfully applied to the analysis of 100 authentic postmortem samples., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Introduction: Cell-free DNA (cfDNA) analysis offers a non-invasive method to identify sensitising and resistance mutations in advanced Non-Small Cell Lung Cancer (NSCLC) patients. Next-generation sequencing (NGS) of circulating free DNA (cfDNA) is a valuable tool for mutations detection and disease's clonal monitoring., Material and Methods: An amplicon-based targeted gene NGS panel was used to analyse 101 plasma samples of advanced non-small cell lung cancer (NSCLC) patients with known oncogenic mutations, mostly EGFR mutations, serially collected at different clinically relevant time points of the disease., Results: The variant allelic frequency (VAF) monitoring in consecutive plasma samples demonstrated different molecular response and progression patterns. The decrease in or the clearance of the mutant alleles was associated with response and the increase in or the emergence of novel alterations with progression. At the best response, the median VAF was 0% (0.0% to 3.62%), lower than that at baseline, with a median of 0.53% (0.0% to 9.9%) ( p = 0.004). At progression, the VAF was significantly higher (median 4.67; range: 0.0-36.9%) than that observed at the best response ( p = 0.001) and baseline ( p = 0.006). These variations anticipated radiographic changes in most cases, with a median time of 0.86 months. Overall, the VAF evolution of different oncogenic mutations predicts clinical outcomes., Conclusion: The targeted NGS of circulating tumour DNA (ctDNA) has clinical utility to monitor treatment response in patients with advanced lung adenocarcinoma.

Moniliophthora perniciosa causes witches' broom disease of cacao and inflicts symptoms suggestive of hormonal imbalance. We investigated whether infection of the tomato (Solanum lycopersicum) model system Micro-Tom (MT) by the Solanaceae (S)-biotype of Moniliophthora perniciosa, which causes stem swelling and hypertrophic growth of axillary shoots, results from changes in host cytokinin metabolism. Inoculation of an MT-transgenic line that overexpresses the Arabidopsis CYTOKININ OXIDASE-2 gene (35S::AtCKX2) resulted in a reduction in disease incidence and stem diameter. RNA-sequencing analysis of infected MT and 35S::AtCKX2 revealed the activation of cytokinin-responsive marker genes when symptoms were conspicuous. The expression of an Moniliophthora perniciosa tRNA-ISOPENTENYL-TRANSFERASE suggests the production of isopentenyladenine (iP), detected in mycelia grown in vitro. Inoculated MT stems showed higher levels of dihydrozeatin and trans-zeatin but not iP. The application of benzyladenine induced symptoms similar to infection, whereas applying the cytokinin receptor inhibitors LGR-991 and PI55 decreased symptoms. Moniliophthora perniciosa produces iP that might contribute to cytokinin synthesis by the host, which results in vascular and cortex enlargement, axillary shoot outgrowth, reduction in root biomass and an increase in fruit locule number. This strategy may be associated with the manipulation of sink establishment to favour infection by the fungus., (© 2021 The Authors New Phytologist © 2021 New Phytologist Foundation.)

Tumor mutation burden (TMB) is evaluated as a biomarker of response to immunotherapy. We present the efforts of the Onconetwork Immuno-Oncology Consortium to validate a commercial targeted sequencing test for TMB calculation. A three-phase study was designed to validate the Oncomine Tumor Mutational Load (OTML) assay at nine European laboratories. Phase 1 evaluated reproducibility and accuracy on seven control samples. In phase 2, six formalin-fixed, paraffin-embedded samples tested with FoundationOne were reanalyzed with the OTML panel to evaluate concordance and reproducibility. Phase 3 involved analysis of 90 colorectal cancer samples with known microsatellite instability (MSI) status to evaluate TMB and MSI association. High reproducibility of TMB was demonstrated among the sites in the first and second phases. Strong correlation was also detected between mean and expected TMB in phase 1 (r 2  = 0.998) and phase 2 (r 2  = 0.96). Detection of actionable mutations was also confirmed. In colorectal cancer samples, the expected pattern of MSI-high/high-TMB and microsatellite stability/low-TMB was present, and gene signatures produced by the panel suggested the presence of a POLE mutation in two samples. The OTML panel demonstrated robustness and reproducibility for TMB evaluation. Results also suggest the possibility of using the panel for mutational signatures and variant detection. Collaborative efforts between academia and companies are crucial to accelerate the translation of new biomarkers into clinical research., (Copyright © 2021 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Artificial intelligence (AI)-based solutions have revolutionized our world, using extensive datasets and computational resources to create automatic tools for complex tasks that, until now, have been performed by humans. Massive data is a fundamental aspect of the most powerful AI-based algorithms. However, for AI-based healthcare solutions, there are several socioeconomic, technical/infrastructural, and most importantly, legal restrictions, which limit the large collection and access of biomedical data, especially medical imaging. To overcome this important limitation, several alternative solutions have been suggested, including transfer learning approaches, generation of artificial data, adoption of blockchain technology, and creation of an infrastructure composed of anonymous and abstract data. However, none of these strategies is currently able to completely solve this challenge. The need to build large datasets that can be used to develop healthcare solutions deserves special attention from the scientific community, clinicians, all the healthcare players, engineers, ethicists, legislators, and society in general. This paper offers an overview of the data limitation in medical predictive models; its impact on the development of healthcare solutions; benefits and barriers of sharing data; and finally, suggests future directions to overcome data limitations in the medical field and enable AI to enhance healthcare. This perspective is dedicated to the technical requirements of the learning models, and it explains the limitation that comes from poor and small datasets in the medical domain and the technical options that try or can solve the problem related to the lack of massive healthcare data.

Background: Analysis of circulating tumor DNA (ctDNA) has remarkable potential as a non-invasive lung cancer molecular diagnostic method. This prospective study addressed the clinical value of a targeted-gene amplicon-based plasma next-generation sequencing (NGS) assay to detect actionable mutations in ctDNA in patients with newly diagnosed advanced lung adenocarcinoma., Methods: ctDNA test performance and concordance with tissue NGS were determined, and the correlation between ctDNA findings, clinical features, and clinical outcomes was evaluated in 115 patients with paired plasma and tissue samples., Results: Targeted-gene NGS-based ctDNA and NGS-based tissue analysis detected 54 and 63 genomic alterations, respectively; 11 patients presented co-mutations, totalizing 66 hotspot mutations detected, 51 on both tissue and plasma, 12 exclusively on tissue, and 3 exclusively on plasma. NGS-based ctDNA revealed a diagnostic performance with 81.0% sensitivity, 95.3% specificity, 94.4% PPV, 83.6% NPV, test accuracy of 88.2%, and Cohen's Kappa 0.764. PFS and OS assessed by both assays did not significantly differ. Detection of ctDNA alterations was statistically associated with metastatic disease ( p = 0.013), extra-thoracic metastasis ( p = 0.004) and the number of organs involved ( p = 0.010)., Conclusions: This study highlights the potential use of ctDNA for mutation detection in newly diagnosed NSCLC patients due to its high accuracy and correlation with clinical outcomes.

Background: Plant pathogenesis related-1 (PR-1) proteins belong to the CAP superfamily and have been characterized as markers of induced defense against pathogens. Moniliophthora perniciosa and Moniliophthora roreri are hemibiotrophic fungi that respectively cause the witches' broom disease and frosty pod rot in Theobroma cacao. Interestingly, a large number of plant PR-1-like genes are present in the genomes of both species and many are up-regulated during the biotrophic interaction. In this study, we investigated the evolution of PR-1 proteins from 22 genomes of Moniliophthora isolates and 16 other Agaricales species, performing genomic investigation, phylogenetic reconstruction, positive selection search and gene expression analysis., Results: Phylogenetic analysis revealed conserved PR-1 genes (PR-1a, b, d, j), shared by many Agaricales saprotrophic species, that have diversified in new PR-1 genes putatively related to pathogenicity in Moniliophthora (PR-1f, g, h, i), as well as in recent specialization cases within M. perniciosa biotypes (PR-1c, k, l) and M. roreri (PR-1n). PR-1 families in Moniliophthora with higher evolutionary rates exhibit induced expression in the biotrophic interaction and positive selection clues, supporting the hypothesis that these proteins accumulated adaptive changes in response to host-pathogen arms race. Furthermore, although previous work showed that MpPR-1 can detoxify plant antifungal compounds in yeast, we found that in the presence of eugenol M. perniciosa differentially expresses only MpPR-1e, k, d, of which two are not linked to pathogenicity, suggesting that detoxification might not be the main function of most MpPR-1., Conclusions: Based on analyses of genomic and expression data, we provided evidence that the evolution of PR-1 in Moniliophthora was adaptive and potentially related to the emergence of the parasitic lifestyle in this genus. Additionally, we also discuss how fungal PR-1 proteins could have adapted from basal conserved functions to possible roles in fungal pathogenesis.

Lung cancer is the leading cause of cancer death worldwide, with non-small cell lung cancer (NSCLC) representing its most commonly diagnosed sub-type. Despite the significant improvements in lung cancer biomarkers knowledge, accompanied by substantial technological advances in molecular tumor profiling, a considerable fraction (up to 30 %) of advanced NSCLC patient presents with major testing challenges or tissue unavailability for molecular analysis. In this context, liquid biopsy is on the rise, currently gaining considerable interest within the molecular pathology and oncology community. Molecular profiling of liquid biopsy specimens using next generation molecular biology methodologies is a rapidly evolving field with promising applications not exclusively limited to advanced stages but also more recently expanding to early stages cancer patients. Here, we offer an overview of some of the most consolidated and emerging applications of next generation sequencing technologies for liquid biopsy testing in NSCLC., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background: Human papillomavirus (HPV) and Trichomonas vaginalis (TV) infections are the most common sexually transmitted infections (STIs) globally. The latter has contributed to a variety of adverse outcomes for both sexes. Moreover, in Brazil, epidemiological studies on patients with STIs are limited. Therefore, this study aimed to determine the prevalence of TV and its association with HPV in women undergoing cervical cancer screening., Methods: Women with a normal cervix were recruited from a community-based cervical cancer screening program. Gynecological examinations were conducted, and questionnaires were provided. Vaginal canal and uterine cervix samples were collected for cytological examinations (reported using the 2001 Bethesda System) and tested for the presence of TV and HPV DNA., Results: In total, 562 women who attended public primary healthcare were included in the study. The T. vaginalis was present in 19.0% (107) and HPV DNA was present in 46.8% (263) of women. Among the women of TV 73.8% (79) had a co-infection with HPV (p = 0.001)., Conclusions: We concluded that a TV infection is associated with an HPV infection of the cervix as well as with the cervical cytological abnormalities. Further studies could reveal the mechanisms by which these two organisms interact at the cellular level, with control for shared behavioral risk factors.

Liquid biopsy is an emerging technology with a potential role in the screening and early detection of lung cancer. Several liquid biopsy-derived biomarkers have been identified and are currently under ongoing investigation. In this article, we review the available data on the use of circulating biomarkers for the early detection of lung cancer, focusing on the circulating tumor cells, circulating cell-free DNA, circulating micro-RNAs, tumor-derived exosomes, and tumor-educated platelets, providing an overview of future potential applicability in the clinical practice. While several biomarkers have shown exciting results, diagnostic performance and clinical applicability is still limited. The combination of different biomarkers, as well as their combination with other diagnostic tools show great promise, although further research is still required to define and validate the role of liquid biopsies in clinical practice., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Freitas, Sousa, Machado, Serino, Santos, Cruz-Martins, Teixeira, Cunha, Pereira, Oliveira, Costa and Hespanhol.)

Melanoma is the deadliest form of skin cancer, primarily due to its high metastatic propensity and therapeutic resistance in advanced stages. The frequent inactivation of the p53 tumour suppressor protein in melanomagenesis may predict promising outcomes for p53 activators in melanoma therapy. Herein, we aimed to investigate the antitumor potential of the p53-activating agent SLMP53-2 against melanoma. Two- and three-dimensional cell cultures and xenograft mouse models were used to unveil the antitumor activity and the underlying molecular mechanism of SLMP53-2 in melanoma. SLMP53-2 inhibited the growth of human melanoma cells in a p53-dependent manner through induction of cell cycle arrest and apoptosis. Notably, SLMP53-2 induced p53 stabilization by disrupting the p53-MDM2 interaction, enhancing p53 transcriptional activity. It also promoted the expression of p53-regulated microRNAs (miRNAs), including miR-145 and miR-23a. Moreover, it displayed anti-invasive and antimigratory properties in melanoma cells by inhibiting the epithelial-to-mesenchymal transition (EMT), angiogenesis and extracellular lactate production. Importantly, SLMP53-2 did not induce resistance in melanoma cells. Additionally, it synergized with vemurafenib, dacarbazine and cisplatin, and resensitized vemurafenib-resistant cells. SLMP53-2 also exhibited antitumor activity in human melanoma xenograft mouse models by repressing cell proliferation and EMT while stimulating apoptosis. This work discloses the p53-activating agent SLMP53-2 which has promising therapeutic potential in advanced melanoma, either as a single agent or in combination therapy. By targeting p53, SLMP53-2 may counteract major features of melanoma aggressiveness.

The abuse of stimulants such as amphetamine, methamphetamine, ecstasy (MDMA), and their analogues (MDEA and MDA) has been increasing considerably worldwide since 2009. In this work, an analytical method using dispersive liquid-liquid microextraction (DLLME) to determine amphetamine and derivatives in oral fluid samples by liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed and validated. Linearity was achieved between 20 to 5000 ng/mL (r>0.992, 1/x² weighted linear regression), with a limit of quantification (LOQ) of 20 ng/mL. Imprecision (%relative standard deviation) and bias (%) were not higher than 9.1 and -12.3%, respectively. The matrix effect was lower than 14.6%, with no carryover observed up to 5000 ng/mL and no interference with 10 different oral fluid matrix sources and against 14 pharmaceuticals and other common drugs of abuse. MDMA, MDA, and MDEA in processed samples were stable up to 24 h at autosampler (10°C); and amphetamine and methamphetamine up to 18 h. The developed method was successfully applied to authentic oral fluid analyses (n = 140). The proposed method is an example of the Green Analytical Toxicology, since it reduces both the amount of solvent required in samples preparation and the quantity of solvents and reagents used in analytical-instrumental stage, as well as requires a minimal sample volume, being a cheaper, quicker and more ecological alternative to conventional methods. Obtained results showed that DLLME extraction combined with LC-MS/MS is a fast and simple method to quantify amphetamine derivatives in oral fluid samples., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Ayahuasca is a beverage obtained from Banisteriopsis caapi plus Psychotria viridis. B. caapi contains the β-carbolines harmine, harmaline, and tetrahydroharmine that are monoamine oxidase inhibitors and P. viridis contains N,N-dimethyltryptamine (DMT) that is responsible for the visionary effects of the beverage. Ayahuasca use is becoming a global phenomenon, and the recreational use of DMT and similar alkaloids has also increased in recent years; such uncontrolled use can lead to severe intoxications. In this investigation, liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to study the kinetics of alkaloids over a 24 h period in saliva and serum of 14 volunteers who consumed ayahuasca twice a month in a religious context. We compared the area under the curve (AUC), maximum concentration (C max ), time to reach C max (T max ), mean residence time (MRT), and half-life (t 1/2 ), as well as the serum/saliva ratios of these parameters. DMT and β-carboline concentrations (C max ) and AUC were higher in saliva than in serum and the MRT was 1.5-3.0 times higher in serum. A generalized estimation equations (GEEs) model suggested that serum concentrations could be predicted by saliva concentrations, despite large individual variability in the saliva and serum alkaloid concentrations. The possibility of using saliva as a biological matrix to detect DMT, β-carbolines, and their derivatives is very interesting because it allows fast noninvasive sample collection and could be useful for detecting similar alkaloids used recreationally that have considerable potential for intoxication., (© 2020 John Wiley & Sons, Ltd.)

Objective: To report a near-fatal poisoning after intentional injection of ricin from a castor bean ( Ricinus communis ) extract., Case Report: A 21 year-old man self-injected ∼3 mL of a castor bean extract intramuscularly and subcutaneously in the left antecubital fossa. Upon admission to our ED (1 h post-exposure; day 1, D1) he was awake and alert, but complained of mild local pain and showed slight local edema and erythema. He evolved to refractory shock (∼24 h post-exposure) that required the administration of a large volume of fluids and high doses of norepinephrine and vasopressin, mainly from D2 to D4. During this period, he developed clinical and laboratory features compatible with systemic inflammatory response syndrome, multiple organ dysfunction, capillary leak syndrome, rhabdomyolysis, necrotizing fasciitis and possible compartment syndrome. The patient underwent forearm fasciotomy on D4 and there was progressive improvement of the hemodynamic status from D7 onwards. Wound management involved several debridements, broad-spectrum antibiotics and two skin grafts. Major laboratory findings within 12 days post-exposure revealed hypoalbuminemia, proteinuria, thrombocytopenia, leukocytosis and increases in cytokines (IL-6, IL-10 and TNF-α), troponin and creatine kinase. Ricin A-chain (ELISA) was detected in serum up to D3 (peak at 24 h post-exposure), with ∼79% being excreted in the urine within 64 h post-exposure. Ricinine was detected in serum and urine by LC-MS up to D5. A ricin A-chain concentration of 246 µg/mL was found in the seed extract, corresponding to the injection of ∼738 µg of ricin A-chain (∼10.5 µg/kg). The patient was discharged on D71, with limited range of motion and function of the left forearm and hand., Conclusion: Ricin injection resulted in a near-fatal poisoning that evolved with septic shock-like syndrome, multiple organ dysfunction and necrotizing fasciitis, all of which were successfully treated with supportive care.

In this study, a comprehensively optimization of QuEChERS (quick, easy, cheap, effective, rugged and safe) method using design of experiments (DOE) was conducted to evaluate the best conditions to obtain the most effective extraction. Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analysis was performed to identify and quantify the antidepressants, with electrospray ionization acquired in positive mode. The method was validated for all analytes; the calibration curves were linear from 10-1000ng/mL, with R 2 >0.98, and with LOD and LOQ defined as 10ng/mL. Method imprecision and bias were less than 14.3% and 18.9%, respectively. Neither carryover nor interferences were observed. Overall, the optimized method was applied in postmortem real sample analysis to quantify the antidepressants. This study showed a viable method that can be applied for routine forensic analysis, with a quick and easy sample preparation and a rapid total run time of 8min for each analysis., Competing Interests: Declaration of Competing Interest The authors report no declarations of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Monensin is an ionophore antibiotic used as a feed additive and growth promoter in cattle production worldwide. The occurrence of monensin in aquatic surficial ecosystems is of concern due to its possible detrimental effects on human health and native biota. Argentina is one of the most important cattle beef producers worldwide; however, there is little knowledge on the environmental occurrence of monensin and the associated risks to aquatic biota. In this study, we developed a method for the extraction and quantification of monensin in surface water; then, we evaluated the occurrence of monensin in a stream impacted by different animal husbandry's operations, and then, we analyzed the ecological implications of monensin residues on aquatic organisms using the risk quotient (RQ) method. Sampling was carried out on August 2017 from the headwaters to the floodplain of the El Pantanoso stream, Buenos Aires province, Argentina. Monensin detection frequency was 75% (n = 20). The median level was 0.40 μg/L and the maximum concentration was 4.70 μg/L. The main input of monensin was from a cattle slaughterhouse, an activity that has not been considered before in the literature as a source of emission of veterinary pharmaceuticals into the environment. The RQ assessment showed that monensin levels could have potential negative effects on aquatic biota in the sampling site closest to the cattle slaughterhouse. The data obtained in this study shows that monensin was present in El Pantanoso surface waters at levels of high ecotoxicological risk to aquatic biota.

Lung cancer is still the leading cause of cancer death in the world. For this reason, novel approaches for early and more accurate diagnosis are needed. Computer-aided decision (CAD) can be an interesting option for a noninvasive tumour characterisation based on thoracic computed tomography (CT) image analysis. Until now, radiomics have been focused on tumour features analysis, and have not considered the information on other lung structures that can have relevant features for tumour genotype classification, especially for epidermal growth factor receptor ( EGFR ), which is the mutation with the most successful targeted therapies. With this perspective paper, we aim to explore a comprehensive analysis of the need to combine the information from tumours with other lung structures for the next generation of CADs, which could create a high impact on targeted therapies and personalised medicine. The forthcoming artificial intelligence (AI)-based approaches for lung cancer assessment should be able to make a holistic analysis, capturing information from pathological processes involved in cancer development. The powerful and interpretable AI models allow us to identify novel biomarkers of cancer development, contributing to new insights about the pathological processes, and making a more accurate diagnosis to help in the treatment plan selection.

Breast cancer is a complex disease whose molecular mechanisms are not completely understood. Developing target therapies is a promising approach. Therefore, understanding the biological behavior of the tumor is a challenge. Tissue biopsy in the metastatic setting remains the standard method for diagnosis. Nevertheless, it has been associated with some disadvantages: It is an invasive procedure, it may not represent tumor heterogeneity, and it does not allow for treatment efficacy to be assessed or early recurrences to be detected. Analysis of circulating tumor DNA (ctDNA) may help to overcome this as it is a non-invasive method of monitoring the disease. In early-stage disease, it can detect early recurrences and monitor tumors' genomic profiles, identifying the emergence of new genetic alterations which can be related to tumor-acquired resistance. In the metastatic setting, the analysis of ctDNA may also allow for the anticipation of clinical and radiological progression of the disease, selection of targeted therapies, and for a photogram of tumor heterogeneity to be provided. It may also detect disease progression earlier in locally advanced tumors submitted to neoadjuvant treatment, and identify minimal residual disease. ctDNA analysis may guide clinical decision-making in different scenarios, in a precision medicine era, once it acts as a repository of genetic tumor material, allowing for a comprehensive mutation profiling analysis. In this review, we focused on recent advances towards the implementation of ctDNA in a clinical routine for breast cancer.

The detection of the markers of Cannabis consumption in biological specimens is an important task for drug testing laboratories in varous contexts. A simple assay combining salting-out assisted liquid-liquid extraction sample preparation and LC-MS/MS analysis was applied to the measurement of Δ 9 -tetrahydrocannabinol, 11-nor-9-carboxy-Δ 9 -tetrahydrocannabinol (THC-COOH), 11-hydroxy-Δ 9 -tetrahydrocannabinol, cannabinol and cannabidiol concentrations in 100 μl plasma specimens. The assay had linearity of 1-100 ng ml -1 for THC-COOH and 0.5-50 ng ml -1 for the other tested cannabinoids. Assay validation criteria were fulfilled. Extraction yields (88.7-97.3%) and internal-standard correct matrix effects (-9.6 to +5.4%) were acceptable. The assay was applied to 238 clinical specimens from trauma patients, with 19 samples presenting quantifiable concentrations of at least one of the target compounds. The developed assay is a simple and efficient strategy for simultaneous measurement of Δ 9 -tetrahydrocannabinol, THC-COOH, 11-hydroxy-Δ 9 -tetrahydrocannabinol, cannabinol and cannabidiol concentrations in plasma specimens., (© 2020 John Wiley & Sons, Ltd.)

An original, selective and automated method, for the microextraction by packed sorbent (MEPS) of Δ9-tetrahydrocannabinol (THC), 11-hydroxy-Δ9-tetrahydrocannabinol (THC-OH), and 11-nor-Δ9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH) from human urine, was developed by using (i) a catechin-molded molecularly imprinted polymer (MIP), (ii) a new lab-made MEPS device easily repackable with any commercial or lab-made sorbent, and (iii) a lab-made multi syringe autosampler. Analyses were performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and the developed method proved to be precise, accurate and showed good linearity. Determination coefficients ranged from 0.96 to 0.99, in the range of 5-250 ng mL -1 . Limits of detection and quantification ranged between 1.0 and 5.0 ng mL -1 and 5.0 and 20.0 ng mL -1 . The method was successfully applied in the analysis of real urine samples. The same packed syringe was effectively used over 90 consecutive extractions without carry-over effects., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Objective: Steroid measurement is a challenge in pediatric endocrinology. Currently, liquid chromatography with tandem mass spectrometry (LC-MS/MS) is considered a gold standard for this purpose. The aim of this study was to compare both LC-MS/MS and immunoassay (IA) for androgens before and after human recombinant chorionic gonadotropin (rhCG) stimulus in children with 46,XY disorders of sex development (DSD)., Methods: Nineteen patients with 46,XY DSD were evaluated; all of them were prepubertal and non-gonadectomized. Testosterone, dihydrotestosterone (DHT), DHEA and androstenedione were measured by IA and LC-MS/MS before and 7 days after rhCG injection. The correlation between IA and LC-MS/MS was analyzed by the intraclass correlation coefficient (ICC) and Spearman's rank correlation coefficient (SCC). For concordance analysis the Passing and Bablok (PB) regression and the Bland and Altman (BA) method were used., Results: Testosterone showed excellent correlation (ICC = 0.960 and SCC = 0.964); DHT showed insignificant and moderate correlations as indicated by ICC (0.222) and SCC (0.631), respectively; DHEA showed moderate correlation (ICC = 0.585 and SCC = 0.716); and androstenedione had poor and moderate correlations in ICC (0.363) and SCC (0.735), respectively. Using the PB method, all hormones showed a linear correlation, but proportional and systematic concordance errors were detected for androstenedione, systematic errors for testosterone and no errors for DHEA and DHT. By the BA method, there was a trend of IA to overestimate testosterone and androstenedione and underestimate DHEA and DHT when compared to LC-MS/MS., Conclusion: Traditional IA should be replaced by LC-MS/MS for the androgens measurement in prepubertal children whenever is possible.