Interplay between liver and blood stages of Plasmodium infection dictates malaria severity via γδ T cells and IL-17-promoted stress erythropoiesis.

Plasmodium replicates within the liver prior to reaching the bloodstream and infecting red blood cells. Because clinical manifestations of malaria only arise during the blood stage of infection, a perception exists that liver infection does not impact disease pathology. By developing a murine model where the liver and blood stages of infection are uncoupled, we showed that the integration of signals from both stages dictated mortality outcomes. This dichotomy relied on liver stage-dependent activation of Vγ4+ γδ T cells. Subsequent blood stage parasite loads dictated their cytokine profiles, where low parasite loads preferentially expanded IL-17-producing γδ T cells. IL-17 drove extra-medullary erythropoiesis and concomitant reticulocytosis, which protected mice from lethal experimental cerebral malaria (ECM). Adoptive transfer of erythroid precursors could rescue mice from ECM. Modeling of γδ T cell dynamics suggests that this protective mechanism may be key for the establishment of naturally acquired malaria immunity among frequently exposed individuals.

Phase 2 Trial of Difelikefalin in Notalgia Paresthetica.

BACKGROUND: Notalgia paresthetica is a neuropathic disorder characterized by pruritus in a circumscribed region of the upper back. Difelikefalin, a selective kappa opioid receptor agonist, has shown efficacy in other chronic pruritic conditions and is being investigated for the treatment of notalgia paresthetica. METHODS: In this phase 2, double-blind, placebo-controlled trial, we randomly assigned, in a 1:1 ratio, patients with moderate-to-severe pruritus caused by notalgia paresthetica to receive 2 mg of oral difelikefalin or placebo twice daily for 8 weeks. The primary outcome was the change from baseline at week 8 in the weekly mean score on the daily Worst Itch Numeric Rating Scale (WI-NRS; scores range from 0 [no itch] to 10 [worst itch imaginable]). The secondary clinical outcomes were itch-related quality-of-life and itch-related sleep measures. RESULTS: A total of 126 patients were enrolled; 62 patients were assigned to receive difelikefalin, and 63 were assigned to receive placebo. One patient who had been assigned to receive difelikefalin withdrew consent before the first dose and is not included in the main analyses. The mean baseline WI-NRS score was 7.6 (indicating severe itch) in each group. The change from baseline in the weekly mean WI-NRS score at week 8 was -4.0 points in the difelikefalin group and -2.4 points in the placebo group (difference in change, -1.6 points; 95% confidence interval, -2.6 to -0.6; P = 0.001). The results for the secondary outcomes generally did not support those of the primary analysis. Headache, dizziness, constipation, and increased urine output occurred more frequently in the difelikefalin group than in the placebo group. CONCLUSIONS: Among patients with notalgia paresthetica, oral treatment with difelikefalin resulted in modestly greater reductions in itch intensity scores than placebo over a period of 8 weeks but was associated with adverse events. Larger and longer trials are needed to assess the efficacy and safety of difelikefalin treatment in this disorder. (Funded by Cara Therapeutics; KOMFORT ClinicalTrials.gov number, NCT04706975.).

ELISL: early-late integrated synthetic lethality prediction in cancer.

MOTIVATION: Anti-cancer therapies based on synthetic lethality (SL) exploit tumour vulnerabilities for treatment with reduced side effects, by targeting a gene that is jointly essential with another whose function is lost. Computational prediction is key to expedite SL screening, yet existing methods are vulnerable to prevalent selection bias in SL data and reliant on cancer or tissue type-specific omics, which can be scarce. Notably, sequence similarity remains underexplored as a proxy for related gene function and joint essentiality. RESULTS: We propose ELISL, Early-Late Integrated SL prediction with forest ensembles, using context-free protein sequence embeddings and context-specific omics from cell lines and tissue. Across eight cancer types, ELISL showed superior robustness to selection bias and recovery of known SL genes, as well as promising cross-cancer predictions. Co-occurring mutations in a BRCA gene and ELISL-predicted pairs from the HH, FGF, WNT, or NEIL gene families were associated with longer patient survival times, revealing therapeutic potential. AVAILABILITY AND IMPLEMENTATION: Data: 10.6084/m9.figshare.23607558 & Code: github.com/joanagoncalveslab/ELISL.

Comparative study of sample preparation procedures to determine the main compounds in ayahuasca beverages by QuEChERS and high-performance liquid chromatography analysis.

INTRODUCTION: Ayahuasca is a psychoactive drink originally consumed by indigenous people of the Amazon. The lack of regulation of this drink leads to uncontrolled consumption, and it is often consumed in religious contexts. OBJECTIVE: The aim of this work is to compare three miniaturised extraction techniques for extracting the main ayahuasca compounds from beverages. METHODOLOGY: Three sample pretreatment techniques were evaluated (dispersive liquid-liquid microextraction [DLLME], microextraction by packed sorbent [MEPS] and QuEChERS [Quick, Easy, Cheap, Effective, Rugged and Safe]) for the simultaneous extraction of N,N-dimethyltryptamine (DMT), tetrahydroharmine (THH), harmine, harmaline, harmol and harmalol from ayahuasca beverage samples. Then, the most promising technique (QuEChERS) was chosen to pre-concentrate the analytes, subsequently detected by high-performance liquid chromatography coupled to a diode array detector (HPLC-DAD). RESULTS: The procedure was optimised, with the final conditions being 500 µL of extractor solvent, 85 mg of primary secondary amine (PSA) and 4 s of vortexing. The analytical method was validated, showing to be linear between 0.16 and 10 µg/mL for ß-carbolines and between 0.016 and 1 µg/mL for DMT, with coefficients of determination (R2) between 0.9968 and 0.9993. The limit of detection (LOD) and lower limit of quantification (LLOQ) were 0.16 µg/mL for all compounds, except for DMT (0.016 µg/mL) and extraction efficiencies varied between 60.2% and 88.0%. CONCLUSION: The analytical methodology proved to be accurate and precise, with good linearity, LODs and LLOQs. This method has been fully validated and successfully applied to ayahuasca beverage samples.

Difelikefalin suppresses itch and reduces scratching independent of inflammation in a murine model of atopic dermatitis.

BACKGROUND: Therapies specifically targeting nonhistaminergic pruritus are largely lacking. Difelikefalin (DFK) has been found to reduce itch in various chronic pruritic conditions, including atopic dermatitis (AD). OBJECTIVE: We sought to investigate the ability of DFK to impact scratching behavior, inflammatory mediators, and neuronal signaling in a murine model of AD. METHODS: The ears of C57BL/6 mice were topically treated with MC903 for 12 consecutive days to induce AD-like inflammation and itch. Before MC903 treatment, mice were treated with either DFK (0.5 mg/kg, intraperitoneal injection twice daily) or vehicle (saline). Skin ear thickness, histological analysis, flow cytometry, RNA-sequencing, and differential gene expression analyses of mouse ear skin were used to examine the effect of DFK on skin inflammation. Scratching behavior was quantified to measure itch behavior in mice that were topically treated with MC903 for 6 consecutive days; then, mice received a single injection of either DFK (1.0 mg/kg, intraperitoneal injection) or saline. Calcium imaging and single-cell RNA-sequencing were used in mouse dorsal root ganglia neurons to determine the size of the neurons activated with DFK treatment. Statistical significance was determined by Mann-Whitney test, unless otherwise noted. RESULTS: DFK rapidly suppressed itch without altering AD-like skin inflammation in MC903 (calcipotriol)-treated mice. In vitro Ca2+ influx trace of dorsal root ganglia suggested that a major target for DFK is the larger-diameter mechanoreceptors (eg, Aꞵ-fibers), rather than small-diameter pruriceptive C-fibers. CONCLUSIONS: These studies support a potential neuromodulatory role of DFK for reducing itch associated with AD in mice.

Oral difelikefalin reduces moderate to severe pruritus and expression of pruritic and inflammatory biomarkers in subjects with atopic dermatitis.

BACKGROUND: Pruritus is the most common and burdensome symptom of atopic dermatitis (AD). Pruritus-targeted treatments in AD are lacking, particularly for patients with milder skin disease. OBJECTIVE: We sought to evaluate the impact of the selective κ-opioid receptor agonist difelikefalin (DFK) on pruritus intensity and pruritus- and immune-related biomarkers in subjects with moderate to severe AD-related pruritus. METHODS: A phase 2 clinical trial investigated the efficacy and safety of oral DFK 0.25, 0.5, and 1.0 mg in subjects with moderate to severe AD-related pruritus. A biomarker substudy evaluated the effects of DFK on the expression of pruritus, TH2-associated genes, and skin barrier-related genes. RESULTS: In the clinical trial (N = 401), all DFK doses reduced itch versus placebo; however, the results were not statistically significant at week 12. In a subgroup of subjects in the trial with mild to moderate skin inflammation and moderate to severe itch (itch-dominant AD phenotype), DFK reduced itch at week 12 versus placebo. In the biomarker substudy, DFK downregulated the expression of key pruritus-related genes (eg, IL-31 and TRPV1) and the AD phenotype (eg, CCL17). Gene set variation analysis confirmed that DFK, but not placebo, downregulated pruritus-related genes and TH2 pathways. DFK improved skin barrier integrity markers and upregulated the expression of claudins and lipid metabolism-associated genes (eg, SEC14L6, ELOVL3, CYP1A2, and AKR1D1). CONCLUSIONS: DFK treatment reduced itch in subjects with moderate to severe AD-related pruritus, particularly those with an "itch-dominant" AD phenotype, and had an impact on the expression of pruritus, TH2-associated genes, and skin barrier-related genes. DFK is a promising therapy for AD-related pruritus; further clinical studies are warranted.

Additive Cytotoxic and Colony-Formation Inhibitory Effects of Aspirin and Metformin on PI3KCA-Mutant Colorectal Cancer Cells.

Human malignancies are one of the major health-related issues throughout the world and are anticipated to rise in the future. Despite huge investments made in anticancer drug development, limited success has been obtained and the average number of FDA approvals per year is declining. So, an increasing interest in drug repurposing exists. Metformin (MET) and aspirin (ASP) possess anticancer properties. This work aims to test the effect of these two drugs in combination on colorectal cancer (CRC) cells in vitro. The effects of MET and/or ASP on cell proliferation, viability, migratory ability, anchorage-independent growth ability (colony formation), and nutrient uptake were determined in two (HT-29 and Caco-2) human CRC cell lines. Individually, MET and ASP possessed antiproliferative, cytotoxic, and antimigratory effects and reduced colony formation in HT-29 cells (BRAF- and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit α (PI3KCA)-mutant), although MET did not affect either 3H-deoxy-D-glucose or 14C-butyrate uptake and lactate production, and ASP caused only a small decrease in 14C-butyrate uptake. Moreover, in these cells, the combination of MET and ASP resulted in a tendency to an increase in the cytotoxic effect and in a potentiation of the inhibitory effect on colony formation, although no additive antiproliferative and antimigratory effects, and no effect on nutrient uptake and lactate production were observed. In contrast, MET and ASP, both individually and in combination, were almost devoid of effects on Caco-2 cells (BRAF- and PI3KCA-wild type). We suggest that inhibition of PI3K is the common mechanism involved in the anti-CRC effect of both MET, ASP and their combination and, therefore, that the combination of MET + ASP may especially benefit PI3KCA-mutant CRC cases, which currently have a poor prognostic.

Overcoming selection bias in synthetic lethality prediction.

MOTIVATION: Synthetic lethality (SL) between two genes occurs when simultaneous loss of function leads to cell death. This holds great promise for developing anti-cancer therapeutics that target synthetic lethal pairs of endogenously disrupted genes. Identifying novel SL relationships through exhaustive experimental screens is challenging, due to the vast number of candidate pairs. Computational SL prediction is therefore sought to identify promising SL gene pairs for further experimentation. However, current SL prediction methods lack consideration for generalizability in the presence of selection bias in SL data. RESULTS: We show that SL data exhibit considerable gene selection bias. Our experiments designed to assess the robustness of SL prediction reveal that models driven by the topology of known SL interactions (e.g. graph, matrix factorization) are especially sensitive to selection bias. We introduce selection bias-resilient synthetic lethality (SBSL) prediction using regularized logistic regression or random forests. Each gene pair is described by 27 molecular features derived from cancer cell line, cancer patient tissue and healthy donor tissue samples. SBSL models are built and tested using approximately 8000 experimentally derived SL pairs across breast, colon, lung and ovarian cancers. Compared to other SL prediction methods, SBSL showed higher predictive performance, better generalizability and robustness to selection bias. Gene dependency, quantifying the essentiality of a gene for cell survival, contributed most to SBSL predictions. Random forests were superior to linear models in the absence of dependency features, highlighting the relevance of mutual exclusivity of somatic mutations, co-expression in healthy tissue and differential expression in tumour samples. AVAILABILITY AND IMPLEMENTATION: https://github.com/joanagoncalveslab/sbsl. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Therapeutic Liquid Eutectic Solvents in Lipid Nanoparticles as Drug Vehicles-A Proof of Concept.

Tuberculosis is an infectious disease caused by the bacterial complex Mycobacterium tuberculosis. Despite the decline in the incidence and mortality of this disease over the years, the emergence of new strains of tuberculosis resistant to existing tuberculostatic drugs is currently one of the largest public health problems. The engineering and development of new drugs is a complex process; therefore, the modification and enhancement of the drugs already marked is a better and faster solution. Ethambutol (ETB) is an antimycobacterial drug used to treat tuberculosis; however, it is highly hygroscopic and is sparingly soluble in water. Therapeutic Deep Eutectic Solvents (THEDESs) are known to improve drug solubility, permeability, and hygroscopicity, among others. In this study, three THEDESs of ETB were synthesized with sucralose, glucose and glycerol and then encapsulated in nanostructured lipid carriers to improve their stability. This work is a proof of concept on the possibility of encapsulating the THEDESs, and results show that the encapsulation of ETB is possible, yielding formulations with a loading capacity superior to 8.5% and able to incorporate THEDESs and not just ETB.

Sex Differences in Tryptophan Metabolism: A Systematic Review Focused on Neuropsychiatric Disorders.

Tryptophan (Tryp) is an essential amino acid and the precursor of several neuroactive compounds within the central nervous system (CNS). Tryp metabolism, the common denominator linking serotonin (5-HT) dysfunctions and neuroinflammation, is involved in several neuropsychiatric conditions, including neurological, neurodevelopmental, neurodegenerative, and psychiatric diseases. Interestingly, most of those conditions occur and progress in a sex-specific manner. Here, we explore the most relevant observations about the influence of biological sex on Tryp metabolism and its possible relation to neuropsychiatric diseases. Consistent evidence suggests that women have a higher susceptibility than men to suffer serotoninergic alterations due to changes in the levels of its precursor Tryp. Indeed, female sex bias in neuropsychiatric diseases is involved in a reduced availability of this amino acid pool and 5-HT synthesis. These changes in Tryp metabolism could lead to sexual dimorphism on the prevalence and severity of some neuropsychiatric disorders. This review identifies gaps in the current state of the art, thus suggesting future research directions. Specifically, there is a need for further research on the impact of diet and sex steroids, both involved in this molecular mechanism as they have been poorly addressed for this topic.

Visual Cortical Plasticity: Molecular Mechanisms as Revealed by Induction Paradigms in Rodents.

Assessing the molecular mechanism of synaptic plasticity in the cortex is vital for identifying potential targets in conditions marked by defective plasticity. In plasticity research, the visual cortex represents a target model for intense investigation, partly due to the availability of different in vivo plasticity-induction protocols. Here, we review two major protocols: ocular-dominance (OD) and cross-modal (CM) plasticity in rodents, highlighting the molecular signaling pathways involved. Each plasticity paradigm has also revealed the contribution of different populations of inhibitory and excitatory neurons at different time points. Since defective synaptic plasticity is common to various neurodevelopmental disorders, the potentially disrupted molecular and circuit alterations are discussed. Finally, new plasticity paradigms are presented, based on recent evidence. Stimulus-selective response potentiation (SRP) is one of the paradigms addressed. These options may provide answers to unsolved neurodevelopmental questions and offer tools to repair plasticity defects.

Evaluation of Antipsychotic Drugs' Stability in Oral Fluid Samples.

Antipsychotics have narrow therapeutic windows, and their monitoring in biological fluids is therefore important; consequently, stability in those fluids must be investigated during method development and validation. This work evaluates the stability of chlorpromazine, levomepromazine, cyamemazine, clozapine, haloperidol, and quetiapine in oral fluid (OF) samples, using the dried saliva spots (DSS) sampling approach and gas chromatography coupled to tandem mass spectrometry. Since many parameters can influence the stability of the target analytes, design of experiments was adopted to check the crucial factors that affect that stability in a multivariate fashion. The studied parameters were the presence of preservatives at different concentrations, temperature, light, and time. It was possible to observe that antipsychotic stability improved when OF samples in DSS were stored at 4 °C, with a low ascorbic acid concentration, and in the absence of light. With these conditions, chlorpromazine and quetiapine were stable for 14 days, clozapine and haloperidol were stable for 28 days, levomepromazine remained stable for 44 days, and cyamemazine was stable for the entire monitored period (146 days). This is the first study that evaluates the stability of these antipsychotics in OF samples after application to DSS cards.

Continuous Production of Dietetic Structured Lipids Using Crude Acidic Olive Pomace Oils.

Crude olive pomace oil (OPO) is a by-product of olive oil extraction. In this study, low-calorie structured triacylglycerols (TAGs) were produced by acidolysis of crude OPO with medium-chain fatty acids (caprylic, C8:0; capric, C10:0) or interesterification with their ethyl ester forms (C8EE, C10EE). These new TAGs present long-chain fatty acids (L) at position sn-2 and medium-chain fatty acids (M) at positions sn-1,3 (MLM). Crude OPO exhibited a high acidity (12.05-28.75% free fatty acids), and high contents of chlorophylls and oxidation products. Reactions were carried out continuously in a packed-bed bioreactor for 70 h, using sn-1,3 regioselective commercial immobilized lipases (Thermomyces lanuginosus lipase, Lipozyme TL IM; and Rhizomucor miehei lipase, Lipozyme RM IM), in solvent-free media at 40 °C. Lipozyme RM IM presented a higher affinity for C10:0 and C10EE. Lipozyme TL IM preferred C10:0 over C8:0 but C8EE over C10EE. Both biocatalysts showed a high activity and operational stability and were not affected by OPO acidity. The New TAG yields ranged 30-60 and the specific productivity ranged 0.96-1.87 g NewTAG/h.g biocatalyst. Lipozyme RM IM cost is more than seven-fold the Lipozyme TL IM cost. Therefore, using Lipozyme TL IM and crude acidic OPO in a continuous bioreactor will contribute to process sustainability for structured lipid production by lowering the cost of the biocatalyst and avoiding oil refining.

A self-comfort oriented pattern of regulatory behavior and avoidant attachment are more likely among infants born moderate-to-late preterm.

Infants born preterm (<37 gestational weeks, GW) are at increased risk for regulatory difficulties and insecure attachment. However, the association between infants' regulatory behavior patterns and their later attachment organization is understudied in the preterm population. We addressed this gap by utilizing a Portuguese sample of 202 mother-infant dyads. Specifically, we compared the regulatory behavior patterns of 74 infants born moderate-to-late preterm (MLPT, 32-36 GW) to those of 128 infants born full-term (FT, 37-42 GW) and evaluated the associations of these regulatory patterns with later attachment. Infants' regulatory behavior patterns (Social-Positive Oriented, Distressed-Inconsolable, or Self-Comfort Oriented) were evaluated in the Face-to-Face-Still-Face paradigm at 3 months, and their attachment organization (secure, insecure-avoidant, or insecure-ambivalent) was evaluated in the Strange Situation at 12 months corrected age. In both samples, the Social-Positive-Oriented regulatory pattern was associated with secure attachment; the Distressed-Inconsolable pattern with insecure-ambivalent attachment; and the Self-Comfort-Oriented pattern with insecure-avoidant attachment. However, compared to FT infants, infants born MLPT were more likely to exhibit a Self-Comfort-Oriented pattern and avoidant attachment. Most perinatal and demographic variables were not related to infant outcomes. However, infants with a higher 1-min Apgar were more likely to exhibit the Social-Positive-Oriented regulatory pattern and secure attachment.

Evaluation of the In Vitro Wound-Healing Potential of Ayahuasca.

Ayahuasca is an Amazonian drink, which contains ß-carboline alkaloids and N,N-dimethyltryptamine. The aim of this study was to evaluate the healing potential of decoctions of a commercial mixture, four individual plants and four mixtures of two plants used in the ayahuasca preparation. Thus, the cytotoxic potential of the samples was evaluated and a wound-healing assay was performed with a NHDF cell line. Subsequently, a parallel artificial membrane permeability assay was also performed, to verify if any psychoactive compound could be absorbed by skin fibroblasts. The integrity and permeability of the cell layer were also evaluated, using the transepithelial electrical resistance assay and Lucifer yellow permeability assay, respectively. The compounds absorbed by the cell layer were quantified by high-performance liquid chromatography coupled to a diode array detector. The results showed that only one sample showed cytotoxicity and all the others promoted the migration of skin fibroblasts. Additionally, it was also verified that ß-carbolynic alkaloids and N,N-dimethyltriptamine were not absorbed by the cell layer, and in general, did not interfere with its permeability and integrity. To the best of our knowledge, this is the first study where ayahuasca's wound-healing potential was evaluated.

Stability of Cocaine, Opiates, and Metabolites in Dried Saliva Spots.

Drug abuse still represents a global problem, and it is associated with an increased risk of diseases, injuries, and deaths. Cocaine (COC) and opiates are the most abused drugs and account for a significant number of fatalities. Therefore, it is important to develop methods capable of effectively identifying and quantifying these substances. The present study aims to evaluate the long-term stability of COC, ecgonine methylester (EME), benzoylecgonine (BEG), cocaethylene (COET), norcocaine (NCOC), morphine (MOR), codeine (COD) and 6-monoacetylmorphine (6-MAM) in oral fluid samples. The analytes of interest were isolated from the matrix (50 µL) using the dried saliva spots (DSS) sampling approach and were subsequently analyzed by gas chromatography coupled with tandem mass spectrometry (GC-MS/MS). The parameters that could influence the stability of the target compounds were studied, and these were storage temperature, light, use of preservatives (and respective concentrations), and time. The effects of each parameter were evaluated using the design of experiments (DOE) approach. The stability of the target analytes was improved when the DSS were stored at room temperature, in the presence of light and using 1% sodium fluoride. The best conditions were then adopted for the DSS storage and long-term stability was assessed. COD was only stable for 1 day, EME was stable for 3 days, COC, COET, NCOC and 6-MAM were stable for 7 days, MOR for 14 days and BEG remained stable throughout the study (136 days). This is the first study that evaluates the stability of these compounds in oral fluid samples after application in DSS cards, and optimizes the conditions in order to improve their stability.

Psychoactive Substances of Natural Origin: Toxicological Aspects, Therapeutic Properties and Analysis in Biological Samples.

The consumption of new psychoactive substances (NPSs) has been increasing, and this problem affects several countries worldwide. There is a class of NPSs of natural origin, consisting of plants and fungi, which have a wide range of alkaloids, responsible for causing relaxing, stimulating or hallucinogenic effects. The consumption of some of these substances is prompted by religious beliefs and cultural reasons, making the legislation very variable or even ambiguous. However, the abusive consumption of these substances can present an enormous risk to the health of the individuals, since their metabolism and effects are not yet fully known. Additionally, NPSs are widely spread over the internet, and their appearance is very fast, which requires the development of sophisticated analytical methodologies, capable of detecting these compounds. Thus, the objective of this work is to review the toxicological aspects, traditional use/therapeutic potential and the analytical methods developed in biological matrices in twelve plant specimens (Areca catechu, Argyreia nervosa, Ayahuasca, Catha edulis, Datura stramonium, Lophophora williamsii, Mandragora officinarum, Mitragyna speciosa, Piper methysticum Forst, Psilocybe, Salvia divinorum and Tabernanthe iboga).

In Vitro Study of the Bioavailability and Bioaccessibility of the Main Compounds Present in Ayahuasca Beverages.

Ayahuasca is a psychoactive beverage that contains the psychoactive compound N,N-dimethyltryptamine and ß-carboline alkaloids. This study aims at determining in vitro the bioavailability and bioaccessibility of the main compounds present in decoctions of four individual plants, in a commercial mixture and in four mixtures of two individual plants used in the preparation of Ayahuasca. The samples were subjected to an in vitro digestion process, and the Caco-2 cell line was used as an absorption model. The integrity and permeability of the cell monolayer were evaluated, as well as the cytotoxicity of the extracts. After digestion and cell incubation, the compounds absorbed by the cell monolayer were quantified by high-performance liquid chromatography coupled to a diode array detector. The results showed that compounds such as N,N-dimethyltryptamine, Harmine, Harmaline, Harmol, Harmalol and Tetrahydroharmine were released from the matrix during the in vitro digestion process, becoming bioaccessible. Similarly, some of these compounds, after being incubated with the cell monolayer, were absorbed, becoming bioavailable. The extracts did not show cytotoxicity after cell incubation, and the integrity and permeability of the cell monolayer were not compromised.

Correction: Simão, A.Y., et al. Evaluation of the Cytotoxicity of Ayahuasca Beverages. Molecules 2020, 25, 5594.

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Pathological stage, surgical margin and lymphovascular invasion as prognostic factors after salvage radiotherapy for post-prostatectomy relapsed prostate cancer - outcomes and optimization strategies.

BACKGROUND: Salvage radiotherapy (sRT) is the main potentially curative treatment after biochemical failure/locoregional relapse post-radical prostatectomy (RP). The aim of the study was to characterize the population who underwent sRT after RP at our Department, to understand the influence of several potential prognosis factors, and to determine possible optimization strategies. MATERIALS AND METHODS: We retrospectively analyzed patients undergoing sRT at our department between 2012 and 2017, evaluating patient, tumor and treatment characteristics, restaging procedures and clinical outcomes - namely biochemical relapse-free survival (BC-RFS), clinical relapse-free survival (C-RFS), additional hormone therapy-free survival (HT-FS) and overall survival (OS). We assessed potential prognostic factors by univariate and multivariate models (MVA). RESULTS: We included 277 patients (median age 68 years). Median pre-sRT PSA was > 0.5ng/mL in 54.9%. All underwent prostate bed irradiation. Pelvic lymph nodes were included in 9.7%. Outcome analysis was performed for 264 patients (35.6 months median follow-up). At 3 years, BC-RFS was 61.4%, C-RFS was 81.3%, HT-FS was 79.9% and OS was 96.6%. Most relapses occurred in regional lymph nodes only (47.9% patients who relapsed). On MVA, lymphovascular invasion, advanced pT-stages and negative margins negatively influenced BC-RFS (p = 0.029, p = 0.002 and p < 0.001) and HT-FS (p = 0.001, p = 0.029 and p = 0.002). C-RFS was worsened by lymphovascular invasion (p = 0.009) and negative margins (p = 0.015). These had no effect on OS. BC-RFS and HT-FS were improved when sRT started while PSA ≤ 0.5 ng/mL (p < 0.05). CONCLUSION: Lymphovascular invasion, higher pT-stages and negative margins negatively affected prognosis. An early start of sRT (PSA ≤ 0.5 ng/mL) predicted better BC-RFS and HT-FS.