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Rapid diagnostic tests (RDTs) significantly impact disease treatment strategy. In Japan, information on the use of RDTs for patients with COVID-19 is limited. Here, we aimed to investigate the RDT implementation rate, pathogen detection rate, and clinical characteristics of patients positive for other pathogens by using COVIREGIJP, a national registry of hospitalized patients with COVID-19.A total of 42,309 COVID-19 patients were included. For immunochromatographic testing, influenza was the most common (n = 2881 [6.8%]), followed by Mycoplasma pneumoniae (n = 2129 [5%]) and group A streptococcus (GAS) (n = 372 [0.9%]). Urine antigen testing was performed for 5524 (13.1%) patients for S. pneumoniae and for 5326 patients (12.6%) for L. pneumophila.The completion rate of M. pneumonia loop-mediated isothermal amplification (LAMP) testing was low (n = 97 [0.2%]). FilmArray RP was performed in 372 (0.9%) patients; 1.2% (36/2881) of patients were positive for influenza, 0.9% (2/223) for the respiratory syncytial virus (RSV), 9.6% (205/2129) for M. pneumoniae, and 7.3% (27/372) for GAS. The positivity rate for urine antigen testing was 3.3% (183/5524) for S. pneumoniae and 0.2% (13/5326) for L. pneumophila. The positivity rate for LAMP test was 5.2% (5/97) for M. pneumoniae. Five of 372 patients (1.3%) had positive FilmArray RP, with human enterovirus being the most frequently detected (1.3%, 5/ 372).The characteristics of patients with and without RDTs submission and positive and negative results differed for each pathogen. RDTs remain an important diagnostic tool in patients with COVID-19 in whom coinfection with other pathogens needs to be tested based on clinical evaluation.

We aimed to compare the viability of circulating polymorphonuclear leukocyte (cPMN) and endometrial PMN (ePMN) and their function dynamics in postpartum dairy cows with subclinical (SCE) or clinical endometritis (CE). To do so, blood samples from 38 Holstein cows were collected at -7, 9, 21, and 36 d relative to calving, and endometrial cytology samples from 32 Holstein cows were harvested at 9, 21, and 36 d postpartum. Uterine health status was assessed at 36 d postpartum, and cows were classified as healthy (absence of abnormal vaginal discharge and =5% ePMN), SCE (absence of abnormal vaginal discharge and >5% ePMN), or CE (mucopurulent or purulent vaginal discharge and >5% ePMN). Viability (viable, apoptotic, and necrotic) and function parameters phagocytosis (PC), oxidative burst, and intracellular proteolytic degradation were evaluated for cPMN via flow cytometry. For ePMN, only viability and PC were evaluated. The association of cPMN and ePMN viability and functional parameters with reproductive tract health classification were fitted in mixed linear regression models, accounting for repeated measures, sampling day, and interactions of reproductive tract status and day. Cows with CE had a lower proportion of cPMN viability (84.5 +/- 2.1%; least squares means +/- standard error) and a higher proportion of apoptosis (14.4 +/- 2.0%) than healthy (92.4 +/- 1.3 and 6.7 +/- 1.3%, respectively) or SCE (95.3 +/- 2.4 and 3.8 +/- 2.3%, respectively) at 9 d postpartum. Interestingly, cPMN intracellular proteolytic degradation was lower [6.2 +/- 0.1 median fluorescence intensity (MFI)] in SCE compared with healthy (6.7 +/- 0.08 MFI) or CE (6.8 +/- 0.1 MFI) at d 9 postpartum. No other differences in cPMN function were found among experimental groups. The proportion of necrotic ePMN was higher for healthy (49.6 +/- 5.1%) than SCE (27.4 +/- 7.3%) and CE (27.7 +/- 7.3%) cows at 36 d postpartum. Also, at 36 d postpartum, the proportion of ePMN performing PC was higher in CE (47.0 +/- 8.6%) than in healthy (18.4 +/- 7.6%) cows, but did not differ from SCE cows (25.9 +/- 8.7%). Results of the present study suggest that cPMN viability and function at 9 d postpartum are associated with the development of uterine disease. Furthermore, ePMN at 36 d postpartum are mostly necrotic in healthy cows but viable and functional in cows with CE, probably due to active uterine inflammation. Remarkably, ePMN in cows with SCE at 36 d postpartum are also mostly viable but seem to display a numerically lower proportion of PC compared with ePMN in CE cows. Key words: transition period, inflammation, immune function, uterine disease

Production of preparations based on natural ingredients has become a priority in the cosmetic and pharmaceutical industries. The novelty of the study was to present a possible method for obtaining pure diacylglycerols (DAG) from natural raw materials like fats (hemp seed oil and mutton tallow). We have determined the optimum water content for enzymatic interesterification to shift the reaction equilibrium towards hydrolysis and obtain the highest DAG formation yield. Then, DAG were separated and purified by column chromatography. Raw fats were characterized by fatty acids composition and their positional distribution using gas chromatography. Triacylglycerols, diacylglycerols, monoacylglycerols and free fatty acid contents were determined using gel permeation chromatography. The main fatty acids found in sn-2 MAG of hemp seed oil was linoleic acid, which was followed by oleic acid. Saturated fatty acids in hemp seed oil’s TAG showed preference for the outer sn-1,3 positions. For mutton tallow, the majority of oleic acid were found at sn-2 position. The optimum water content applied during enzymatic interesterification for DAG formation was estimated to be 2.0 % w/w, using the following conditions: 5 % w/w of catalyst, 200 rpm, 60 °C, 6h. DAG yield was 27 %. Purification of the mutton tallow-based and hemp seed oil-based DAG yielded with 83 % w/w and 82 % w/w, respectively. High purity DAG could be applied as effective emulsifiers in food, cosmetic or pharmaceutical emulsions.

IntroductionAntibiotics represent the most common type of medication used during pregnancy and infancy. Antibiotics have been proposed as a possible factor in changes in microbiota composition, which may play a role in the aetiology of autism and attention deficit/hyperactivity disorder (ADHD). Our aim was to investigate the association between maternal and early-life antibiotic use and autism and ADHD in childhood.MethodsThis Swedish nation-wide population-based cohort study included all first live singleton births (N = 483,459) between January 2006 and December 2016. The association of dispensed antibiotics with autism and ADHD in children aged = 1 antibiotic during the exposure period (from 3 months pre-conception to delivery), and 41.6% (n = 201,040) of the children received >= 1 antibiotic in early life (aged

Selegiline is an irreversible, selective type-B monoamine oxidase inhibitor (MAOI) approved for Parkison's disease-oral and major depressive disorder-transdermal formulation) result-ing in non-selective MAOI activity at oral doses >20 mg/day. The present systematic re-view and meta-analysis appraises the evidence of different formulations/dosages of selegiline across different psychiatric conditions. We inquired PubMed/MEDLINE/Cochrane-Central/WHO-ICTRP/Clarivate-WebOfScience and the Chinese-Electronic-Journal Database from inception to 10/26/2022 for selegiline trials involving psychiatric patients. Random-effects meta-analyses assessed heterogeneity, publication/risk biases, and confidence in the evidence, followed by sensitivity, subgroup, and meta-regression analyses. Co-primary outcomes were: changes in symptom score (standardized mean difference =SMD) and author-defined response (risk ratios =RRs). RRs of adverse events and all-cause discontinuation were secondary and accept-ability outcomes, respectively. Systematic-review included 42 studies; meta-analysis, 23. Se-legiline outperformed placebo in depressive symptom reduction (SMD=-0.96, 95%C.I.=-1.78,-0.14, k = 10, n = 1,308), depression (RR = 1.61, 95%C.I. = 1.20, 2.15, k = 9, n = 1,238) and atypical-depression response (RR = 2.23, 95%C.I. = 1.35, 3.68, k = 3, n = 136). Selegiline failed to outperform the placebo in negative ( k = 4) or positive symptoms of schizophrenia ( k = 4), attention-deficit-hyperactivity disorder (ADHD) symptoms reduction ( k = 2), and smoking ab-stinence rate ( k = 4). Selegiline did not differ from methylphenidate and ADHD scores ( k = 2). No significant difference emerged in acceptability, incident diarrhea, headache, dizziness, and nausea RRs, in contrast to xerostomia (RR = 1.58, 95%C.I. = 1.03, 2.43, k = 6, n = 1,134), insom-nia (RR = 1.61, 95%C.I. = 1.19, 2.17, k = 10, n = 1,768), and application-site reaction for trans -dermal formulation (RR = 1.81, 95%C.I. = 1.40, 2.33, k = 6, n = 1,662). Confidence in findings was low/very-low for most outcomes; moderate for depressive symptoms reduction (transder-mal). Selegiline proved effective, safe, and well-tolerated for depressive disorders, yet further evidence is warranted about specific psychiatric disorders.(c) 2023 Elsevier B.V. and ECNP. All rights reserved.

Despite the high prevalence of alcoholic liver disease, its identification and characterization remain poor, especially in early stages such as alcoholic fatty liver disease and alcoholic steatohepatitis. This latter implies diagnostic difficulties, few therapeutic options and unclear mechanisms of action. To elucidate the metabolic alterations and pinpoint affected biochemical pathways, alcoholic steatohepatitis was simulated in vitro by exposing HepaRG cells to ethanol (IC10, 368 mM) and tumor necrosis factor alpha (TNF-α, 50 ng/mL) for 24 h. This combined exposure was compared to solely ethanol-exposed as well as -nonexposed cells. Four different metabolomics platforms were used combining liquid chromatography, high-resolution mass spectrometry and drift tube ion mobility to elucidate both intracellular and extracellular metabolic alterations. Some of the key findings include the influence of TNF-α in the upregulation of hepatic triglycerides and the downregulation of hepatic phosphatidylethanolamines and phosphatidylcholines. S-Adenosylmethionine showed to play a central role in the progression of alcoholic steatohepatitis. In addition, fatty acyl esters of hydroxy fatty acid (FAHFA)-containing triglycerides were detected for the first time in human hepatocytes and their alterations showed a potentially important role during the progression of alcoholic steatohepatitis. Ethoxylated phosphorylcholine was identified as a potential new biomarker of ethanol exposure.

Context. Off-label, compassionate use of potential drugs against SARS-CoV-2 acute infection could modify their safety profiles. Aims. To evaluate the prevalence and type of adverse drug reactions (ADRs), together with associated risk factors, among Cuban COVID-19 patients treated with chloroquine (CQ), lopinavir/ritonavir (LPV/r), or interferon α2b (IFN α2b), according to the Cuban protocol. Materials and Methods. A prospective descriptive analysis of ADRs was performed on 200 COVID-19 patients who were admitted consecutively to three hospitals in Havana and Pinar del Río from April to July 2020. Information on demographics, ADRs, outcomes, behavioral, and health-related factors was collected using a validated questionnaire and clinical records. Each potential ADR case was assessed for causality based on the WHO-UMC algorithm, concomitant drug influences, and the presence of any drug-drug interactions (DDI). Results. The total frequency of ADRs was 55%, with predominantly gastrointestinal disorders and general symptoms (23% vs 20%). 95.1% of ADRs occurred within 10 days after treatment and 42 potential DDI in 55.5% of patients (61/110) were observed. The prevalence of ADRs was: 44%, 30.4%, and 26.4% for IFN α2b, LPV/r, and CQ, respectively. Sex (odds ratio (OR): 0.40 (95% confidence interval (CI): 0.211–0.742), age (OR: 2.36 (95% CI: 1.02–5.44)), and underlying diseases (OR: 0.12 (95% CI: 0.06–0.23)) were independently associated factors for ADRs ( P < 0.05 ). Conclusions. The frequency of ADRs and potential DDI was high compared to their use during nonpandemic times (e.g., for malaria, HIV, or inflammatory diseases). The safety profile of these drugs when used for COVID-19 treatment showed similar characteristics. Comorbidities, age >37 years old, and female sex were associated with ADRs.

Intracellular delivery is critical for a plethora of biomedical applications, including mRNA transfection and gene editing. High transfection efficiency and low cytotoxicity, however, are often beyond the capabilities of bulk techniques and synonymous with extensive empirical optimization. Moreover, bulk techniques are not amenable to large screening applications. Here, we propose an expeditious workflow for achieving optimal electroporation-based intracellular delivery. Using the multiplexing ability of a high-definition microelectrode array (MEA) chip, we performed a sequence of carefully designed experiments, multiple linear regression modelling and validation to obtain optimal conditions for on-chip electroporation of primary fibroblasts. Five electric pulse parameters were varied to generate 32 different electroporation conditions. The effect of the parameters on cytotoxicity and intracellular delivery could be evaluated with just two experiments. Most successful electroporation conditions resulted in no cell death, highlighting the low cytotoxicity of on-chip electroporation. The resulting delivery models were then used to achieve dosage-controlled delivery of small molecules, delivery of Cas9-GFP single-guide RNA complexes and transfection with an mCherry-encoding mRNA, resulting in previously unreported high-efficiency, single-cell transfection on MEAs: cells expressed mCherry on 81% of the actuated electrodes, underscoring the vast potential of CMOS MEA technology for the transfection of primary cells.

In the present review, we synthesize information on the mechanisms of chronic copper (Cu) toxicity using an adverse outcome pathway framework and identify three primary pathways for chronic Cu toxicity: disruption of sodium homeostasis, effects on bioenergetics, and oxidative stress. Unlike acute Cu toxicity, disruption of sodium homeostasis is not a driving mechanism of chronic toxicity, but compensatory responses in this pathway contribute to effects on organism bioenergetics. Effects on bioenergetics clearly contribute to chronic Cu toxicity with impacts at multiple lower levels of biological organization. However, quantitatively translating these impacts into effects on apical endpoints such as growth, amphibian metamorphosis, and reproduction remains elusive and requires further study. Copper-induced oxidative stress occurs in most tissues of aquatic vertebrates and is clearly a significant driver of chronic Cu toxicity. Although antioxidant responses and capacities differ among tissues, there is no clear indication that specific tissues are more sensitive than others to oxidative stress. Oxidative stress leads to increased apoptosis and cellular damage in multiple tissues, including some that contribute to bioenergetic effects. This also includes oxidative damage to tissues involved in neuroendocrine axes and this damage likely alters the normal function of these tissues. Importantly, Cu-induced changes in hormone concentrations and gene expression in endocrine-mediated pathways such as reproductive steroidogenesis and amphibian metamorphosis are likely the result of oxidative stress-induced tissue damage and not endocrine disruption. Overall, we conclude that oxidative stress is likely the primary driver of chronic Cu toxicity in aquatic vertebrates, with bioenergetic effects and compensatory response to disruption of sodium homeostasis contributing to some degree to observed effects on apical endpoints. Environ Toxicol Chem 2022;00:1-18. (c) 2022 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Toxicological data on overdose with human immunodeficiency virus inhibitors are scarce. We present a case report of two independent suicide attempts by self-administered overdose with the same antiretroviral medicine Genvoya® (emtricitabine/elvitegravir/tenofovir alafenamide/cobicistat). Both patients were admitted to the hospital and presented with a loss of consciousness, lactic acidosis, elevated hepatic transaminase levels and hemodynamic instability. While one patient survived with advanced supportive measures, the other passed away. Emtricitabine levels were measured in vivo in various consecutive serum samples and postmortem urine, peripheral and cardiac serum samples and confirmed excessive use in both cases. This is the first time that emtricitabine levels following overdose are reported. Although measured concentrations for emtricitabine were quite similar in these cases, metabolic acidosis was more pronounced in the fatal case. The difference in outcomes between the two could be due to a difference in physiological status, susceptibility to accumulation and adverse effects, and perhaps a varying interval between ingestion and the start of supportive measures.

Background: The fruit pulp decoction of Crescentia cujete, commonly known as calabash, is traditionally used for the treatment of several respiratory diseases and is available as syrup formulations. Unfortunately, there is no detailed investigation on analytical methods for warranting the quality of these products. Aim and objective: To develop and validate an appropriate analytical method for the simultaneous quantification of transcinnamic acid, 4-hydroxybenzoic acid, verbascoside and 6-epi-aucubin in the decoction and commercial cough syrups of Crescentia cujete fruit. Materials and methods: A reversed-phase ultra-high-performance liquid chromatographic method coupled to a diode array detector (UPLC-DAD) was validated following the ICH guidelines. The chromatographic analysis was performed using a C18 column, the mobile phase system consisted of water and acetonitrile containing 0.1% formic acid, and UV chromatograms were recorded from 200 to 400 nm. Results: A new UPLC-DAD method was validated for the simultaneous quantification of trans-cinnamic acid, 4- hydroxybenzoic acid, verbascoside and 6-epi-aucubin in calabash-derived products. After successful validation, this method was applied for the quantification of the selected chemical markers in an in-house decoction and three commercial cough syrups. Among the selected chemical markers, 6-epi-aucubin was the main compound in the calabash decoction, while trans-cinnamic acid and 4-hydroxybenzoic acid were the major compounds in the commercial products. Verbascoside and 6-epi-aucubin were below the limit of quantification in all syrup samples. Conclusion: The proposed method was successfully applied for the analysis of three commercial syrup formulations and can be useful for standardization and quality control of raw and pharmaceutical calabash preparations.

Current test strategies to identify thyroid hormone (TH) system disruptors are inadequate for conducting robust chemical risk assessment required for regulation. The tests rely heavily on histopathological changes in rodent thyroid glands or measuring changes in systemic TH levels, but they lack specific new approach methodologies (NAMs) that can adequately detect TH-mediated effects. Such alternative test methods are needed to infer a causal relationship between molecular initiating events and adverse outcomes such as perturbed brain development. Although some NAMs that are relevant for TH system disruption are available–and are currently in the process of regulatory validation–there is still a need to develop more extensive alternative test batteries to cover the range of potential key events along the causal pathway between initial chemical disruption and adverse outcomes in humans. This project, funded under the Partnership for the Assessment of Risk from Chemicals (PARC) initiative, aims to facilitate the development of NAMs that are specific for TH system disruption by characterizing in vivo mechanisms of action that can be targeted by in embryo/in vitro/in silico/in chemico testing strategies. We will develop and improve human-relevant in vitro test systems to capture effects on important areas of the TH system. Furthermore, we will elaborate on important species differences in TH system disruption by incorporating non-mammalian vertebrate test species alongside classical laboratory rat species and human-derived in vitro assays.

In Mycobacterium tuberculosis, bedaquiline and clofazimine resistance occurs primarily through Rv0678 variants, a gene encoding a repressor protein that regulates mmpS5/mmpL5 efflux pump gene expression. Despite the shared effect of both drugs on efflux, little else is known about other pathways affected. We hypothesized that in vitro generation of bedaquiline- or clofazimine-resistant mutants could provide insight into additional mechanisms of action. We performed whole-genome sequencing and determined phenotypic MICs for both drugs on progenitor and mutant progenies. Mutants were induced through serial passage on increasing concentrations of bedaquiline or clofazimine. Rv0678 variants were identified in both clofazimine- and bedaquiline-resistant mutants, with concurrent atpE SNPs occurring in the latter. Of concern was the acquisition of variants in the F420 biosynthesis pathway in clofazimine-resistant mutants obtained from either a fully susceptible (fbiD: del555GCT) or rifampicin mono-resistant (fbiA: 283delTG and T862C) progenitor. The acquisition of these variants possibly implicates a shared pathway between clofazimine and nitroimidazoles. Pathways associated with drug tolerance and persistence, F420 biosynthesis, glycerol uptake and metabolism, efflux, and NADH homeostasis appear to be affected following exposure to these drugs. Shared genes affected by both drugs include Rv0678, glpK, nuoG, and uvrD1. Genes with variants in the bedaquiline resistant mutants included atpE, fadE28, truA, mmpL5, glnH, and pks8, while clofazimine-resistant mutants displayed ppsD, fbiA, fbiD, mutT3, fadE18, Rv0988, and Rv2082 variants. These results show the importance of epistatic mechanisms as a means of responding to drug pressure and highlight the complexity of resistance acquisition in M. tuberculosis. In Mycobacterium tuberculosis, bedaquiline and clofazimine resistance occurs primarily through Rv0678 variants, a gene encoding a repressor protein that regulates mmpS5/mmpL5 efflux pump gene expression. Despite the shared effect of both drugs on efflux, little else is known about other pathways affected.

The Amaryllidaceae species are well-known as a rich source of bioactive compounds in nature. Although Hymenocallis littoralis has been studied for decades, its polar components were rarely explored. The current phytochemical investigation of Amaryllidaceae alkaloids from H. littoralis led to the identification of three previously undescribed compounds: O-demethyl-norlycoramine (1), (-)-2-epi-pseudolycorine (2) and (+)-2-epi-pseudolycorine (3), together with eight known compounds: 6α-hydroxyhippeastidine (4), 6β-hydroxyhippeastidine (5), lycorine (6), 2-epi-lycorine (7), zephyranthine (8), ungeremine (9), pancratistatin (10) and 9-O-demethyl-7-O-methyllycorenine (11). Among the eight previously reported compounds, five were isolated from H. littoralis for the first time (compounds 4, 5, 7, 8, and 9). Compounds 1, 4, 5, 7, 8, and 11 exhibited weak anti-SARS-CoV-2 activity (EC50 = 40-77 µM) at non-cytotoxic concentrations. Assessment of cytotoxicity on the Vero-E6 cell line revealed lycorine and pancratistatin as cytotoxic substances with CC50 values of 1.2 µM and 0.13 µM, respectively. The preliminary structure-activity relationship for the lycorine-type alkaloids in this study was further investigated, and as a result ring C appears to play a crucial role in their anti-SARS-CoV-2 activity. ispartof: MOLECULES vol:28 issue:7 ispartof: location:Switzerland status: published

Aims Many clinical decision support systems trigger warning alerts for drug-drug interactions potentially leading to QT prolongation and torsades de pointes (QT-DDIs). Unfortunately, there is overalerting and underalerting because stratification is only based on a fixed QT-DDI severity level. We aimed to improve QT-DDI alerting by developing and validating a risk prediction model considering patient- and drug-related factors. Methods We fitted 31 predictor candidates to a stepwise linear regression for 1000 bootstrap samples and selected the predictors present in 95% of the 1000 models. A final linear regression model with those variables was fitted on the original development sample (350 QT-DDIs). This model was validated on an external dataset (143 QT-DDIs). Both true QTc and predicted QTc were stratified into three risk levels (low, moderate and high). Stratification of QT-DDIs could be appropriate (predicted risk = true risk), acceptable (one risk level difference) or inappropriate (two risk levels difference). Results The final model included 11 predictors with the three most important being use of antiarrhythmics, age and baseline QTc. Comparing current practice to the prediction model, appropriate stratification increased significantly from 37% to 54% appropriate QT-DDIs (increase of 17.5% on average [95% CI +5.4% to +29.6%], padj = 0.006) and inappropriate stratification decreased significantly from 13% to 1% inappropriate QT-DDIs (decrease of 11.2% on average [95% CI −17.7% to −4.7%], padj ≤ 0.001). Conclusion The prediction model including patient- and drug-related factors outperformed QT alerting based on QT-DDI severity alone and therefore is a promising strategy to improve DDI alerting.

Background Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors known to regulate glucose and fatty acid metabolism, inflammation, endothelial function and fibrosis. PPAR isoforms have been extensively studied in metabolic diseases, including type 2 diabetes and cardiovascular diseases. Recent data extend the key role of PPARs to liver diseases coursing with vascular dysfunction, including nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH). Aim This review summarises and discusses the pathobiological role of PPARs in cardiovascular diseases with a special focus on their impact and therapeutic potential in NAFLD and NASH. Results and Conclusions PPARs may be attractive for the treatment of NASH due to their liver-specific effects but also because of their efficacy in improving cardiovascular outcomes, which may later impact liver disease. Assessment of cardiovascular disease in the context of NASH trials is, therefore, of the utmost importance, both from a safety and efficacy perspective.

The glycopeptide vancomycin has an antimicrobial effect by halting the synthesis of the bacterial cell-wall by binding the D-Ala-D-Ala peptide motif of the cell-wall precursor Lipid II. This interaction has been extensively investigated using techniques such as XRD and NMR. Here, vancomycin complexed with Ala-γGlu-Lys-D-Ala-D-Ala (the pentapeptide part of Lipid II) is studied by means of Raman and Raman optical activity (ROA) spectroscopy. Where for conventional structural elucidation techniques, the direct study of a drug-biological target complex is routine, it is not the case for the ROA technique. Through experimental recordings and DFT-level spectral calculations of the complex, it was found that ROA specifically probes the conformation of vancomycin, even in the presence of the biological target. As such, it could be determined that no noticeable conformational changes are involved when binding Ala-γGlu-Lys-D-Ala-D-Ala. This work showcases the interest of directly examining interacting systems conformationally by means of ROA, potentially complementing and/or confirming findings from other studies, and eventually contributing to a more complete understanding of the structure–activity relationship of a drug compound.

Many studies report negative associations between exposure to large concentrations of contaminants and life-history traits of animals. As a consequence, they argue to derive linear models that can be applied to predict effects in sub-threshold or lowdose exposure scenarios. However, several recent studies found that exposure to low doses of a contaminant may induce a hormetic response, i.e. stimulatory or beneficial effects at low concentrations, but harmful effects at higher concentrations. Hormesis might be a novel fundamental pillar in the field of ecotoxicology, as it may promote the evolution of adaptive coping mechanisms in rapidly changing and challenging environments. However, because hormesis occurs in the lowdose zone of the dose-response, it has been often neglected. In this review, we have discussed how low-dose contaminant exposure may adaptively modify molecular and physiological mechanisms that may lead to organisms better capable of coping with challenging environments, how such responses may be transgenerational, and how thus this issue can no longer be ignored in ecotoxicological studies. In so doing, we have also identified some of the main limitations of ecotoxicological studies that, by masking potential hormetic responses of animals to chemicals, make the integration of hormesis complicated to achieve.

Objectives: In order to elaborate a new national challenge panel of resistant Gram-negative bacilli and Gram-positive cocci strains for the validation of routine antimicrobial susceptibility testing (AST) methods, an interlaboratory evaluation was organised. Methods: The results of 12 well-characterised multidrug-resistant strains tested by nine laboratories using local disk diffusion (DD) and automated AST (AUST) methods were compared with the reference broth microdilution method. Results: Overall categorical agreement ranged from 70% to 100% both for DD and AUST and was >90% for all but one strain for all antibiotics. Conclusion: Our multicentre AST study showed good reproducibility and the panel can be used as national resistant reference strains for routine AST validation. (C) 2022 The Author(s). Published by Elsevier Ltd on behalf of International Society for Antimicrobial Chemotherapy. The Belgian national reference centre is supported in part by the Belgian Ministry of Social Affairs through a fund within the national health insurance system (INAMI-RIZIV). The authors thank the members of the Belgian National Antimicrobial Susceptibility Testing Committee (NAC): Olivier Denis, Stefanie Desmet, Youri Glupczynski, Marie Hallin, Te-Din Huang, Bénédicte Lissoir, Koen Magerman, Veerle Matheeussen, Pierrette Melin, Karl Mertens, Hector Rodriguez, Anne-Marie Van Den Abeele and Kris Vernelen. The authors also thank Audric Deckers for his help in compiling the statistics for this article.

Three plasticizers, namely bis (3,5,5-trimethylhexyl) phosphate (TMHPh), di(propylene glycol) dibenzoate (DiPGDB), and tri-n-butyl trimellitate (TBTM), were recently identified and reported in high concentrations in indoor dust from Belgian homes. In this study, their behavior within the human body was investigated by generating Phase I biotransformation products for the first time. Human liver microsomes (HLMs) were used following an in vitro assay and liquid chromatography time of flight mass spectrometry (LC-QTOF-MS) was employed for the analysis. Biotransformation products were identified for TMHPh as products of hydroxylation reactions that took place in one or two positions in the structure of the substrate. For DiPGDB, biotransformation products were formed after hydrolysis of carboxylic esters and oxidative-O-dealkylation. For TBTM, biotransformation products were formed through hydrolysis of the different carboxylic esters of the molecule, in agreement with studies on structurally similar compounds. The generated results can contribute to biomonitoring studies creating new knowledge on human exposure to emerging compounds and on the metabolism of xenobiotics.

Type II NADH dehydrogenase (NDH-2) is an essential component of electron transfer in many microbial pathogens but has remained largely unexplored as a potential drug target. Previously, quinolinyl pyrimidines were shown to inhibit Mycobacterium tuberculosis NDH-2, as well as the growth of the bacteria [Shirude, P. S.; ACS Med. Chem. Lett. 2012, 3, 736−740]. Here, we synthesized a number of novel quinolinyl pyrimidines and investigated their properties. In terms of inhibition of the NDH-2 enzymes from M. tuberculosis and Mycobacterium smegmatis, the best compounds were of similar potency to previously reported inhibitors of the same class (half-maximal inhibitory concentration (IC50) values in the low-μM range). However, a number of the compounds had much better activity against Gram-negative pathogens, with minimum inhibitory concentrations (MICs) as low as 2 μg/mL. Multivariate analyses (partial least-squares (PLS) and principle component analysis (PCA)) showed that overall ligand charge was one of the most important factors in determining antibacterial activity, with patterns that varied depending on the particular bacterial species. In some cases (e.g., mycobacteria), there was a clear correlation between the IC50 values and the observed MICs, while in other instances, no such correlation was evident. When tested against a panel of protozoan parasites, the compounds failed to show activity that was not linked to cytotoxicity. Further, a strong correlation between hydrophobicity (estimated as clog P) and cytotoxicity was revealed; more hydrophobic analogues were more cytotoxic. By contrast, antibacterial MIC values and cytotoxicity were not well correlated, suggesting that the quinolinyl pyrimidines can be optimized further as antimicrobial agents. Lu Lu, Linda Åkerbladh and Shabbir Ahmad contributed equally to this work

Background: Covid-19 vaccination has started in the majority of the countries at the global level. Cancer patients are at high risk for infection, serious illness, and death from COVID-19 and need vaccination guidance and support. Guidance availability in the English language only is a major limit for recommendations’ delivery and their application in the world’s population and generates information inequalities across the different populations. Methods: Most of the available COVID-19 vaccination guidance for cancer patients was screened and scrutinized by the European Cancer Patients Coalition (ECPC) and an international oncology panel of 52 physicians from 33 countries. Results: A summary guidance was developed and provided in 28 languages in order to reach more than 70 percent of the global population. Conclusion: Language barrier and e-guidance availability in the native language are the most important barriers when communicating with patients. E-guidance availability in various native languages should be considered a major priority by international medical and health organizations that are communicating with patients at the global level.

Background: Inappropriate use of clonazepam by the elderly patients is associated with cognitive impairment, delirium and falls. Strategies to optimize its use are important to increase patient safety. Objective: To evaluate the feasibility of a clonazepam deprescription protocol in the elderly. Methods: This is a quasi-experimental study. Elderly people with chronic use of clonazepam and attended in primary care units in two Brazilian municipalities were selected. A deprescription protocol was used, which included five fortnightly meetings between the older adults and the research team, to reduce the dose by 25 %. Patients received instructions on sleep hygiene behaviors and the advantages of clonazepam deprescription; family physicians followed a flowchart for gradual dose reduction. In the 1st and 5th meetings, there were medical appointments for anamnesis and discharge. The monitoring of patients and the application of tests were carried out by the research team. Results: Of the 35 elderly people included in the study, 27 reached the end; 81.5 % achieved deprescription: 22.2 % stopped completely and 59.3 % decreased the dose. At the last meeting, 20 % of elderly patients reported an increase in blood pressure. Conclusions: The high rate of deprescription and the little relevance of clonazepam withdrawal reactions, showed that the use of the protocol was effective. However, the increase in blood pressure and the worsening of sleep quality in the last meeting show the need for adjustment in the last stage of the deprescription process.Trial Registration: The Brazilian Registry of Clinical Trials (ReBEC) RBR-524ys9; registered June 10, 2019.

Background Edible insects are a nutritious and sustainable alternative source of animal proteins, and consequently are nowadays promoted as human food and animal feed worldwide. Ensuring the quality, safety, and traceability of insect foods is thus critical to maintain a safe and economically sustainable supply chain. Providing a simultaneous analysis of a wide range of chemical compounds, metabolomics and non-target screening (NTS) have become important mass spectrometry-based tools in the field of food sciences. At this moment, however, the application of these analytical techniques to edible insect-related topics has been largely overlooked. Scope and approach In this Commentary, we explore the potential of “entometabolomics” for food science and industry. We address the hurdles associated with safety, quality, and traceability of insects as food and feed and current issues related with insect industrial applications. Key findings and conclusions Current applications of metabolomics and NTS in traditional food systems are presented. We propose ways to translate and implement such techniques for the edible insect sector. Even if the broad applicability of metabolomics and NTS approaches is still hampered by technical challenges, we show the high potential of entometabolomics when implemented into the insect food and feed industry and research.

Black soldier fly (BSF) larvae (Hermetia illucens) are voracious feeders that can be reared on food waste streams originating from the food industry and retailers. Because these food waste streams are automatically being unpacked in substantial amounts, they can contain microplastics, potentially jeopardising the larvae’s chemical safety when applied as compound feed ingredients. During this study, the dynamics of ingestion and excretion of microplastics by BSF larvae reared on substrates containing different contents (wMP = 0.00, 0.01, 0.10, 0.50, 1.00, 3.00%) of fluorescent blue-labelled microplastics (median size, Dv(50) = 61.5 µm) were monitored. To correlate the particle size with their uptake, larval mouth opening dimensions were measured during the rearing process. In conclusion, it appeared that ingestion of microplastics by BSF larvae depends on initial particle load, mouth size, and consequently also age. The larvae took up between 131 (wMP = 0.01%) and 4866 (wMP = 3.00%) particles leading to bioaccumulation factors (BAF) between 0.12 (wMP = 3.00%) and 1.07 (wMP = 0.01%). Larvae also appeared to excrete the microplastics, lowering the BAFs to values between 0.01 (wMP = 3.00%) and 0.54 (wMP = 0.01%).

Up to 80% of antibiotics are prescribed in the community. An assessment of prescribing by indication will help to identify areas where improvement can be made. A point prevalence audit study (PPAS) of consecutive respiratory tract infection (RTI) consultations in general practices in 13 European countries was conducted in January–February 2020 (PPAS-1) and again in 2022 (PPAS-4). The European Surveillance of Antibiotic Consumption quality indicators (ESAC-QI) were calculated to identify where improvements can be made. A total of 3618 consultations were recorded for PPAS-1 and 2655 in PPAS-4. Bacterial aetiology was suspected in 26% (PPAS-1) and 12% (PPAS-4), and an antibiotic was prescribed in 30% (PPAS-1) and 16% (PPAS-4) of consultations. The percentage of adult patients with bronchitis who receive an antibiotic should, according to the ESAC-QI, not exceed 30%, which was not met by participating practices in any country except Denmark and Spain. For patients (≥1) with acute upper RTI, less than 20% should be prescribed an antibiotic, which was achieved by general practices in most countries, except Ireland (both PPAS), Croatia (PPAS-1), and Greece (PPAS-4) where prescribing for acute or chronic sinusitis (0–20%) was also exceeded. For pneumonia in adults, prescribing is acceptable for 90–100%, and this is lower in most countries. Prescribing for tonsillitis (≥1) exceeded the ESAC-QI (0–20%) in all countries and was 69% (PPAS-1) and 75% (PPAS-4). In conclusion, ESAC-QI applied to PPAS outcomes allows us to evaluate appropriate antibiotic prescribing by indication and benchmark general practices and countries. Up to 80% of antibiotics are prescribed in the community. An assessment of prescribing by indication will help to identify areas where improvement can be made. A point prevalence audit study (PPAS) of consecutive respiratory tract infection (RTI) consultations in general practices in 13 European countries was conducted in January–February 2020 (PPAS-1) and again in 2022 (PPAS-4). The European Surveillance of Antibiotic Consumption quality indicators (ESAC-QI) were calculated to identify where improvements can be made. A total of 3618 consultations were recorded for PPAS-1 and 2655 in PPAS-4. Bacterial aetiology was suspected in 26% (PPAS-1) and 12% (PPAS-4), and an antibiotic was prescribed in 30% (PPAS-1) and 16% (PPAS-4) of consultations. The percentage of adult patients with bronchitis who receive an antibiotic should, according to the ESAC-QI, not exceed 30%, which was not met by participating practices in any country except Denmark and Spain. For patients (≥1) with acute upper RTI, less than 20% should be prescribed an antibiotic, which was achieved by general practices in most countries, except Ireland (both PPAS), Croatia (PPAS-1), and Greece (PPAS-4) where prescribing for acute or chronic sinusitis (0–20%) was also exceeded. For pneumonia in adults, prescribing is acceptable for 90–100%, and this is lower in most countries. Prescribing for tonsillitis (≥1) exceeded the ESAC-QI (0–20%) in all countries and was 69% (PPAS-1) and 75% (PPAS-4). In conclusion, ESAC-QI applied to PPAS outcomes allows us to evaluate appropriate antibiotic prescribing by indication and benchmark general practices and countries.

Xiphidium caeruleum Aubl. is traditionally used in Cuba as an analgesic, anti-inflammatory, antilithiatic and diuretic remedy. Here we studied the pharmacognostic parameters of the leaves of X. caeruleum, the preliminary phytochemical composition, diuretic activity and acute oral toxicity of the aqueous extracts from the leaves of plants collected in the vegetative (VE) and flowering (FE) stages. The morphological characteristics and physicochemical parameters of leaves and extracts were determined. The phytochemical composition was assessed by phytochemical screening, TLC, UV, IR and HPLC/DAD profiles. The diuretic activity was evaluated in Wistar rats and compared to furosemide, hydrochlorothiazide and spironolactone. Epidermal cells, stomata and crystals were observed on the leaf surface. Phenolic compounds were identified as the main metabolites, including phenolic acids (gallic, caffeic, ferulic and cinnamic acids) and flavonoids (catechin, kaempferol-3-O-glucoside and quercetin). VE and FE showed diuretic activity. The activity of VE was similar to furosemide, and the activity of FE resembled spironolactone. No acute oral toxicity was observed. The presence of flavonoids and phenols in VE and FE may explain at least in part the traditional use and provide some insight into the reported ethnomedical use as a diuretic. Because of the differences in polyphenol profiles between VE and FE, further studies should be carried out to standardize the harvesting and extraction conditions in order to use X. caeruleum leaf extract as herbal medicine.

Hanseniaspora opuntiae is a commonly found yeast species in naturally fermenting cocoa pulp-bean mass, which needed in-depth investigation. The present study aimed at examining effects of the cocoa isolate H. opuntiae IMDO 040108 as part of three different starter culture mixtures compared with spontaneous fermentation, regarding microbial community, substrate consumption, and metabolite production dynamics, including volatile organic compound (VOC) and phytochemical compositions, as well as compositions of the cocoa beans after fermentation, cocoa liquors, and chocolates. The inoculated H. opuntiae strain was unable to prevail over background yeasts present in the fermenting cocoa pulp-bean mass. It led to under-fermented cocoa beans after four days of fermentation, which was however reflected in higher levels of polyphenols. Cocoa fermentation processes inoculated with a Saccharomyces cerevisiae strain enhanced flavour production during the fermentation and drying steps, which was reflected in richer and more reproducible aroma profiles of the cocoa liquors and chocolates. Sensory analysis of the cocoa liquors and chocolates further demonstrated that S. cerevisiae led to more acidic notes compared to spontaneous fermentation, as a result of an advanced fermentation degree. Finally, different VOC profiles were found in the cocoa beans throughout the whole chocolate production chain, depending on the fermentation process.

Activity-based probes (ABP) are molecules that bind covalently to the active form of an enzyme family, making them an attractive tool for target and biomarker identification and drug discovery. The present study describes the synthesis and biochemical characterization of novel activity-based probes targeting trypsin-like serine proteases. We developed an extensive library of activity-based probes with "clickable" affinity tags and a diaryl phosphonate warhead. A wide diversity was achieved by including natural amino acid analogs as well as basic polar residues as side chains. A detailed enzymatic characterization was performed in a panel of trypsin-like serine proteases. Their inhibitory potencies and kinetic profile were examined, and their IC50 values, mechanism of inhibition, and kinetic constants were determined. The activity-based probes with a benzyl guanidine side chain showed the highest inhibitory effects in the panel. Surprisingly, some of the high-affinity probes presented a reversible inhibitory mechanism. On the other hand, probes with different side chains exhibited the expected irreversible mechanism. For the first time, we demonstrate that not only irreversible probes but also reversible probes can tightly label recombinant proteases and proteases released from human mast cells. Even under denaturing SDS-PAGE conditions, reversible slow-tight-binding probes can label proteases due to the formation of high-affinity complexes and slow dissociation rates. This unexpected finding will transform the view on the required irreversible nature of activity-based probes. The diversity of this library of activity-based probes combined with a detailed enzyme kinetic characterization will advance their applications in proteomic studies and drug discovery. ispartof: FRONTIERS IN CHEMISTRY vol:10 ispartof: location:Switzerland status: published

Lactic acid bacteria (LAB) form a group of bacteria to which most probiotics belong and are commonly found in fermented dairy products. Fermented foods and beverages are foods made through desired microbial growth and enzymatic conversions of food components. In this study, 43 LAB were isolated from Ethiopian traditional cottage cheese, cheese, and yogurt and evaluated for their functional and safety properties as candidate probiotics. Twenty-seven isolates, representative of each fermented food type, were selected and identified to the species level. Limosilactobacillus fermentum was found to be the predominant species in all samples studied (70.4%), while 11.1% of isolates were identified as Lactiplantibacillus plantarum. All 27 isolates tested showed resistance to 0.5% bile salt, while 26 strains were resistant to pH 3. The LAB isolates were also evaluated for antagonistic properties against key pathogens, with strain-specific features observed for their antimicrobial activity. Five strains from cottage cheese (Lactiplantibacillus plantarum 54B, 54C, and 55A, Lactiplantibacillus pentosus 55B, and Pediococcus pentosaceus 95E) showed inhibitory activity against indicator pathogens that are key causes of gastrointestinal infections in Ethiopia, i.e., Escherichia coli, Salmonella enterica subsp. enterica var. Typhimurium, Staphylococcus aureus, Shigella flexneri, and Listeria monocytogenes. Strain-specific immunomodulatory activity monitored as nuclear factor kappa B (NF-κB) and interferon regulatory factor (IRF) activation was documented for Lactiplantibacillus plantarum 54B, 55A and P. pentosaceus 95E. Antibiotic susceptibility testing confirmed that all LAB isolates were safe concerning their antibiotic resistance profiles. Five isolates (especially Lactiplantibacillus plantarum 54B, 54C, and 55A, Lactiplantibacillus pentosus 55B, and P. pentosaceus 95E) showed promising results in all assays and are novel probiotic candidates of interest for clinical trial follow-up.

Abstract: Immediate hypersensitivity reactions can pose a clinical and diagnostic challenge, mainly because of the multifarious clinical presentation and distinct underlying – frequently uncertain – mechanisms. Anaphylaxis encompasses all rapidly developing and life-threatening signs and may cause death. Evidence has accumulated that immediate hypersensitivity and anaphylaxis do not necessarily involve an allergen-specific immune response with cross-linking of specific IgE (sIgE) antibodies bound to their high-affinity IgE receptor (FcεRI) on the surface of mast cells (MCs) and basophils. Immediate hypersensitive and anaphylaxis can also result from alternative specific and nonspecific MC and basophils activation and degranulation, such as complementderived anaphylatoxins and off-target occupancy of MC and/or basophil surface receptors such as the Masrelated G protein-coupled receptor X2 (MRGPRX2). Degranulation of MCs and basophils results in the release of inflammatory mediators, which can be, depending on the underlying trigger, in a different spatiotemporal manner. In addition, hypersensitivity and anaphylaxis can occur entirely independently of MC and basophil degranulation, as observed in hypersensitivity to nonsteroidal anti-inflammatory drugs (NSAIDs) that divert normal arachidonic acid metabolism by inhibiting the cyclooxygenase (COX)-1 isoenzyme. Finally, one should remember that anaphylaxis might be part of the phenotype of particular - sometimes poorly recognizable - conditions such as clonal MC diseases (e.g. mastocytosis) and MC activation syndrome (MCAS). This review provides a status update on the molecular mechanisms involved in both sIgE/FcεRI- and non-sIgE/FcεRIdependent immediate hypersensitivity and anaphylaxis. In conclusion, there is increasing evidence for alternative pathophysiological hypersensitivity and anaphylaxis endotypes that are phenotypically and biologically indistinguishable, which are frequently difficult to diagnose, mainly because of uncertainties associated with diagnostic tests that might not enable to unveil the underlying mechanism.

BackgroundCD70-CD27 is a costimulatory ligand-receptor pair in the tumor necrosis factor receptor family. With only limited expression in normal tissues, CD70 is constitutively expressed in a variety of solid tumors and hematologic malignancies, facilitating immunosuppression through CD27 signaling in the tumor microenvironment by enhanced survival of regulatory T cells, induction of T cell apoptosis, and T cell exhaustion. Consequently, CD70 is an increasingly recognized target for developing antibody-based therapies, but its expression patterns vary among different tumor types in spatial distribution, magnitude of expression and percentage of positive cells. In that regard, individual confirmation of CD70 expression at screening and during treatment could enhance the successful implementation of anti-CD70 therapies. Here, we developed a gallium-68 (Ga-68) radiolabeled single-domain antibody-fragment targeting CD70 for in vivo positron emission tomography (PET) imaging.ResultsAn anti-CD70 VHH construct containing a C-direct-tag with a free thiol was developed to enable site-specific conjugation to a NOTA bifunctional chelator for Ga-68 radiolabeling. [Ga-68]Ga-NOTA-anti-CD70 VHH was obtained in good radiochemical yield of 30.4 +/- 1.7% and high radiochemical purity (> 94%). The radiolabeled VHH showed excellent in vitro and in vivo stability. Specific binding of [Ga-68]Ga-NOTA-anti-CD70 VHH was observed on CD70(high) 786-O cells, showing significantly higher cell-associated activity when compared to the blocking condition (p < 0.0001) and CD70(low) NCl-H1975 cells (p < 0.0001). PET imaging showed specific radiotracer accumulation in CD70 expressing human tumor xenografts, which was efficiently blocked by prior injection of unlabeled anti-CD70 VHH (p = 0.0029). In addition, radiotracer uptake in CD70(high) tumors was significantly higher when compared with CD70(low) tumors (p < 0.0001). The distribution of the radioactivity in the tumors using autoradiography was spatially matched with immunohistochemistry analysis of CD70 expression.Conclusion[Ga-68]Ga-NOTA-anti-CD70 VHH showed excellent in vivo targeting of CD70 in human cancer xenografts. PET imaging using this radioimmunoconjugate holds promise as a non-invasive method to identify and longitudinally follow-up patients who will benefit most from anti-CD70 therapies.

Small molecule structure elucidation using tandem mass spectrometry (MS/MS) plays a crucial role in life science, bioanalytical, and pharmaceutical research. There is a pressing need for increased throughput of compound identification and transformation of historical data into information-rich spectral databases. Meanwhile, molecular networking, a recent bioinformatic framework, provides global displays and system-level understanding of complex LC-MS/MS data sets. Herein we present meRgeION, a multifunctional, modular, and flexible R-based toolbox to streamline spectral database building, automated structural elucidation, and molecular networking. The toolbox offers diverse tuning parameters and the possibility to combine various algorithms in the same pipeline. As an open-source R package, meRgeION is ideally suited for building spectral databases and molecular networks from privacy-sensitive and preliminary data. Using meRgeION, we have created an integrated spectral database covering diverse pharmaceutical compounds that was successfully applied to annotate drug-related metabolites from a published nontargeted metabolomics data set as well as reveal the chemical space behind this complex data set through molecular networking. Moreover, the meRgeION-based processing workflow has demonstrated the usefulness of a spectral library search and molecular networking for pharmaceutical forced degradation studies. meRgeION is freely available at: https://github.com/daniellyz/meRgeION2.

Since its discovery 20 years ago, many studies have been performed to gain insight into the functional role of cytoglobin (Cygb). However, Cygb has been proven to be a promiscuous protein. Yet, there is a consensus that Cygb is a cytoprotective protein involved in redox homeostasis. CYGB is a ubiquitously expressed hexacoordinated globin that is highly expressed in melanocytes and is often found to be downregulated during melanocyte-to-melanoma transition. In Chapter III, we investigated the molecular mechanism through which CYGB could be involved in redox regulation. Here, we showed that CYGB contains two redox-sensitive cysteine residues and that the formation of an intramolecular disulfide bridge resulted in the heme group becoming more accessible to external ligands. This supports the hypothesis that Cys38 and Cys83 serve as sensitive redox sensors. In Chapter IV we showed that CYGB mRNA and protein levels were elevated upon exposure to hypoxia. Interestingly, this upregulation was most likely HIF-2α-dependent. We propose that in melanoma, HIF-2α, rather than HIF-1α, positively regulates CYGB under hypoxic conditions in a cell type specific way. In Chapter V, the cytotoxic effect of indirect NTP treatment in two melanoma cell lines with divergent endogenous CYGB expression levels was investigated. We confirmed that NTP endows cytotoxicity that induces cell death through apoptosis and that this was mediated through the production of ROS. Moreover, we showed that CYGB protects melanoma cells from ROS-induced apoptosis by the scavenging of ROS. Interestingly, CYGB expression influenced the expression of NRF2 and HO-1. We identified the lncRNA MEG3 as a possible mechanism through which NRF2 expression and its downstream target HO-1 can be regulated by CYGB. In chapter VI, increased basal ROS levels and higher degree of lipid peroxidation upon RSL3 treatment contributed to the increased sensitivity of CYGB knockdown G361 cells to ferroptosis. Furthermore, transcriptome analysis demonstrates the enrichment of multiple cancer malignancy pathways upon CYGB knockdown, supporting a tumor-suppressive role for CYGB. Remarkably, CYGB expression regulation was identified as a critical determinant of the ferroptosis–pyroptosis therapy response. This suggests that CYGB is involved in the regulation of multiple modes of programmed cell death. FInally, we sought to delineate the RONS that are responsible for plasma-induced ICD. Our results highlight the importance of the short-lived species. Furthermore, we are first to demonstrate that NTP-created vaccine is safely prepared and offers complete protection. Moreover, we provide conclusive evidence that direct application of NTP induces ICD in melanoma.

The Democratic Republic of the Congo has faced fifteen Ebola virus disease (EVD) outbreaks, among which the 2018-2020 was the most widespread and deadliest so far. During that outbreak associated with chronic insecurity, community mistrust and resistance, thirteen field laboratories were deployed to support the EVD diagnosis with the GeneXpert. As this latter cannot be used everywhere in remote areas due to its additional requirements, rapid diagnostic tests (RDTs) were proposed as an alternative tool to support quick decision-making at the point-of-care. However, questions regarding RDTs diagnostic performance and usability were raised due to their disparate performances. In the first part of this PhD thesis, we showed how decentralized and strategically positioned diagnostic laboratories quickly helped to mitigate the risk of Ebola virus spread through rapid, efficient, accurate and well-structured response. Quick hand over of competences and capacities to local teams led to successful management of further health emergencies (EVD flare-ups and Covid-19 pandemic), as those laboratories had dedicated equipment and well-trained local personnel. Sequencing data guided public health decision-making, helped understanding the outbreak dynamics, at risk populations and exposed health zones. In the second part of this PhD, QuickNaviTM RDT had high specificity and quite good sensitivity than OraQuick and Coris tests in outbreaks conditions. QuickNavi-EbolaTM was less impressive compared to previous studies; OraQuick test was almost in line with previous findings, although it performed better for the middle and lower Ct-values in laboratory conditions. None of the four Ebola RDTs evaluated throughout our studies, achieved the desired (sensitivity >98%, specificity >99%) or acceptable (sensitivity >95%, specificity >99%) levels of performance as stated by the WHO Target Product Profile for EBOV tests. However, respective specificities of the QuickNaviTM (>99%) and OraQuick Ebola (98%) in most our studies were close to the acceptable level of performance (>99%). Based on overall performances, QuickNaviTM and Oraquick Ebola RDTs were proposed as a screening panel at the point-of-care to triage and isolate suspect-cases waiting for the RT-qPCR results. RDTs results expectancy will be done separately i.e. individuals with at least one positive RDT isolated in the high-risk area and those with negative RDTs results in low-risk area. For postmortem surveillance, OraQuick RDT effectively complemented the response efforts, improved the community engagement, and decreased the number of systematic safe and dignified burials (SDBs) in corpses with non-reactive test. Trust towards postmortem testing led families to voluntarily request for SDBs despite OraQuick non-reactive results.

Ex vivo modification of T cells with exogenous cargo is a common prerequisite for the development of T cell therapies, such as chimeric antigen receptor therapy. Despite the clinical success and FDA approval of several such products, T cell manufacturing presents unique challenges related to therapeutic efficacy after adoptive cell transfer and several drawbacks of viral transduction-based manufacturing, such as high cost and safety concerns. To generate cellular products with optimal potency, engraftment potential and persistence in vivo, recent studies have shown that minimally differentiated T cell phenotypes are preferred. However, genetic engineering of quiescent T cells remains challenging. Photoporation is an upcoming alternative non-viral transfection method which makes use of photothermal nanoparticles, such as polydopamine nanoparticles (PDNPs), to induce transient membrane permeabilization by distinct photothermal effects upon laser irradiation, allowing exogenous molecules to enter cells. In this study, we analyzed the capability of PDNP-photoporation to deliver large model macromolecules (FITC-dextran 500 kDa, FD500) in unstimulated and expanded human T cells. We compared different sizes of PDNPs (150, 250 and 400 nm), concentrations of PDNPs and laser fluences and found an optimal condition that generated high delivery yields of FD500 in both T cell phenotypes. A multiparametric analysis of cell proliferation, surface activation markers and cytokine production, revealed that unstimulated T cells photoporated with 150 nm and 250 nm PDNPs retained their propensity to become activated, whereas those photoporated with 400 nm PDNPs did less. Our findings show that PDNP-photoporation is a promising strategy for transfection of quiescent T cells, but that PDNPs should be small enough to avoid excessive cell damage.

Proteases catalyze the hydrolysis of peptides or proteins by cleaving peptide bonds. These enzymes are of great interest in biomedical research due to their involvement in many physiological processes. In this thesis, we focus on serine proteases. Serine proteases are involved in several physiological processes, including immune response, cell death, and tissue healing. The upregulation of these proteases can increase inflammatory cytokines, degradation of extracellular matrix components, among others. We previously obtained an in vivo proof of concept with a multi-target serine protease inhibitor (UAMC-00050) in Dry Eye Disease (DED) and Irritable Bowel Syndrome (IBS). Topical application of this compound in the eye of a tear-deficient dry eye rat animal model reduced both tissue damage and inflammatory parameters. Moreover, UAMC-00050 also causes a decrease in visceral hypersensitivity in a rat model of post-inflammatory visceral hypersensitivity. The focus of this project was to characterize the proteases involved in DED and IBS. We wanted to use a proteomic technique, activity-based protein profiling (ABPP). ABPP uses chemical probes, known as activity-based probes (ABPs), which react covalently with the active form of the target enzyme. These allow detection, visualization, or affinity purification of the labeled enzymes. During this project, we developed an extensive library of ABPs with “clickable” affinity tags and a diaryl phosphonate warhead. A wide diversity was achieved by including natural amino acid analogs as well as basic polar residues as side chains. A detailed enzymatic characterization was performed in a panel of trypsin-like serine proteases. Their inhibitory potencies and kinetic profile were examined, and their IC50 values, mechanism of inhibition, and kinetic constants were determined. Surprisingly, some of the high-affinity probes presented a reversible inhibitory mechanism. For the first time, we demonstrated that not only irreversible probes but also reversible probes can tightly label recombinant proteases and proteases released from human mast cells. Even under denaturing SDS-PAGE conditions, reversible slow-tight-binding probes can label proteases due to the formation of high-affinity complexes and slow dissociation rates. In further studies, we aim to identify upregulated proteases in DED and IBS biological samples. Finally, given the promising in vitro results of the ABPs with recombinant enzymes, we developed two new inhibitors targeting trypsin-like serine proteases. The results of their inhibitory potencies for a panel of trypsin-like serine proteases and the first study of their physicochemical properties showed a promising backup compound with the potential to be a therapeutic agent for DED and IBS.

IntroductionMajor depressive disorder remains a major challenge due to its biopsychosocial burden with increased morbidity and mortality. Despite successful treatment options for the acute episode, recurrence rates are high, on average four times in a life span.Areas coveredBoth pharmacological as non-pharmacological evidence-based therapeutic options to prevent and treat recurrent depression are discussed.Expert opinionAlthough some risk factors for recurrence are well known, better evidence is needed. Antidepressant medication should be continued after acute treatment at its full therapeutic dose for longer periods, at least 1 year. There are no clear differences between classes of antidepressant medication when treatment is focused on preventing relapse. Bupropion is the only antidepressant with a proven efficacy to prevent recurrence in seasonal affective disorder. Recent findings conclude maintenance subanesthetic ketamine and esketamine treatment can be effective in sustaining antidepressant effect following remission. Furthermore, the pharmacological approach must be integrated with lifestyle interventions, especially aerobic exercise. Finally, combining pharma- and psychotherapy seems to improve outcome. Network and complexity sciences will help to decrease the high recurrence rates of MDD by developing more integrative and personalized approaches.

(1) Background: Filipendula ulmaria (L.) Maxim. (Rosaceae) (meadowsweet) is widely used in phytotherapy against inflammatory diseases. However, its active constituents are not exactly known. Moreover, it contains many constituents, such as flavonoid glycosides, which are not absorbed, but metabolized in the colon by gut microbiota, producing potentially active metabolites that can be absorbed. The aim of this study was to characterize the active constituents or metabolites. (2) Methods: A F. ulmaria extract was processed in an in vitro gastrointestinal biotransformation model, and the metabolites were characterized using UHPLC-ESI-QTOF-MS analysis. In vitro anti-inflammatory activity was evaluated by testing the inhibition of NF-κB activation, COX-1 and COX-2 enzyme inhibition. (3) Results: The simulation of gastrointestinal biotransformation showed a decrease in the relative abundance of glycosylated flavonoids such as rutin, spiraeoside and isoquercitrin in the colon compartment, and an increase in aglycons such as quercetin, apigenin, naringenin and kaempferol. The genuine as well as the metabolized extract showed a better inhibition of the COX-1 enzyme as compared to COX-2. A mix of aglycons present after biotransformation showed a significant inhibition of COX-1. (4) Conclusions: The anti-inflammatory activity of F. ulmaria may be explained by an additive or synergistic effect of genuine constituents and metabolites.

Leishmania is a protozoan parasite causing a neglected tropical disease known as leishmaniasis, which is fatal in visceral infections if untreated. Few drugs are available for treatment and the emergence of drug resistance poses a threat to current elimination efforts. Leishmania has a plastic genome characterized by dynamic modulation of chromosomes copy number (aneuploidy), which leads to different karyotypes co-existing in clonal populations (mosaic aneuploidy). It is hypothesized that Leishmania exploits mosaic aneuploidy as a strategy for early adaptation to environmental stresses, in particular to drug pressure. In this thesis, we aimed to investigate the extent and dynamics of mosaic aneuploidy during adaptation to standard in vitro conditions as well as to drugs. We first applied a high throughput single-cell genome sequencing (SCGS) to reveal for the first time the complete karyotype of thousands of individual Leishmania parasites in two distinct clonal populations in standard in vitro culture. We observed that drastic changes in karyotypes quickly emerge in a population stemming from an almost euploid cell, with mosaic aneuploidy further increasing by moderate and gradual karyotypic alterations. We also found that all chromosomes are prone to somy changes, but only some polysomies can achieve high frequencies in the population, suggesting that natural selection determines which karyotypes emerge and propagate. In a second set of experiments, we investigated the dynamics of mosaic aneuploidy during adaptation to high drug pressure (SbIII and miltefosine) in vitro. By combining SCGS with lineage tracing using cellular barcodes and longitudinal genome characterization, we revealed that early aneuploidy changes observed under SbIII pressure result from the polyclonal selection of pre-existing karyotypes, complemented by rapid cumulative karyotypic alterations which culminates in similar aneuploidy modifications, pointing to a process of convergent evolution. In the case of miltefosine, early parasite adaptation was associated with independent pre-existing point mutations in a miltefosine transporter gene and aneuploidy changes only emerged later, upon exposure to increased concentration of the drug. Different mutants of the miltefosine transporter gene were identified, also pointing out the polyclonal origin of miltefosine resistance. In summary, the present thesis demonstrates that polyclonality and genome plasticity are hallmarks of Leishmania adaptation, with the scenario of aneuploidy dynamics being dependent on the nature and strength of the environmental stress as well as on the existence of other pre-adaptive mechanisms.

Despite considerable progress in understanding drug metabolism in the human pediatric population, data remains scarce in preterm neonates. Improving our knowledge of the ADME properties in this vulnerable age group is of utmost importance to avoid suboptimal dosing, which may lead to adverse drug reactions. The juvenile (mini)pig is a representative model for hepatic drug metabolism in human neonates and infants, especially phase I reactions. However, the effect of prematurity on the onset of hepatic phase I and phase II enzyme activity has yet to be investigated in this animal model. Therefore, the aim of this study was to assess the ontogeny of CYP3A and UGT enzyme activity in the liver of preterm (gestational day 105–107) and term-born (gestational day 115–117) domestic piglets. In addition, the ontogeny pattern between the preterm and term group was compared to examine whether postconceptional or postnatal age affects the onset of enzyme activity. The following age groups were included: preterm postnatal day (PND) 0 (n = 10), PND 5 (n = 10), PND 11 (n = 8), PND 26 (n = 10) and term PND 0 (n = 10), PND 5 (n = 10), PND 11 (n = 8), PND 19 (n = 18) and PND 26 (n = 10). Liver microsomes were extracted, and the metabolism of CYP3A and UGT-specific substrates assessed enzyme activity. Preterm CYP3A activity was only detectable at PND 26, whereas term CYP3A activity showed a gradual postnatal increase from PND 11 onwards. UGT activity gradually increased between PND 0 and PND 26 in preterm and term-born piglets, albeit, being systematically lower in the preterm group. Thus, postconceptional age is suggested as the main driver affecting porcine CYP3A and UGT enzyme ontogeny. These data are a valuable step forward in the characterization of the preterm piglet as a translational model for hepatic drug metabolism in the preterm human neonate.

The proliferation of new psychoactive substances (NPS) over recent years has made their surveillance complex. The analysis of raw municipal influent wastewater can allow a broader insight into community consumption patterns of NPS. This study examines data from an international wastewater surveillance program that collected and analysed influent wastewater samples from up to 47 sites in 16 countries between 2019 and 2022. Influent wastewater samples were collected over the New Year period and analysed using validated liquid chromatography – mass spectrometry methods. Over the three years, a total of 18 NPS were found in at least one site. Synthetic cathinones were the most found class followed by phenethylamines and designer benzodiazepines. Furthermore, two ketamine analogues, one plant based NPS (mitragynine) and methiopropamine were also quantified across the three years. This work demonstrates that NPS are used across different continents and countries with the use of some more evident in particular regions. For example, mitragynine has highest mass loads in sites in the United States, while eutylone and 3-methylmethcathinone increased considerably in New Zealand and in several European countries, respectively. Moreover, 2F-deschloroketamine, an analogue of ketamine, has emerged more recently and could be quantified in several sites, including one in China, where it is considered as one of the drugs of most concern. Finally, some NPS were detected in specific regions during the initial sampling campaigns and spread to additional sites by the third campaign. Hence, wastewater surveillance can provide an insight into temporal and spatial trends of NPS use., The authors sincerely thank the wastewater treatment plant operators for their help in collecting samples and the support provided by the lab staff who performed the extraction of the samples. The authors also thank SCIEX for the use of their instrument and Liam O'Brien for his statistical advice. The University of Iceland wants to particularly acknowledge Efla hf. and Veitur ohf. for their assistance with sample collection. SLU acknowledges the support of Inga Haalck, Oscar G¨otlind and Gabriel Persson for sample collection. IGE acknowledges the support of Nelly Laforet and Myriam Millet-Perrin for sample collection. The Queensland Alliance for Environmental Health Sciences, The University of Queensland, gratefully acknowledges the financial support of the Queensland Department of Health. R. B. is the recipient of an Australian Research Council Discovery Early Career Award (project number DE220100381), and J. M. is the recipient of an Australian Research Council Australian Laureate Fellowship (project number FL200100028) funded by the Australian Government. N.R acknowledges funding from the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska–Curie Grant Agreement No. 896141. L. B. acknowledges the grant RYC2020–028936-I funded by MCIN/AEI/ 10.13039/501100011033 and by "ESF Investing in your future". E.G-L. acknowledges the financial support of the Madrid Government (Comunidad de Madrid- Spain) under the Multiannual Agreement with Complutense University in the line Program to Stimulate Research for Young Doctors in the context of the V PRICIT (Regional Programme of Research and Technological Innovation) (project number PR65/19–22432). T.B, A.C. and A. v. N. would like to thank European Union's Justice Programme—Drugs Policy Initiatives, EuSeME (project number: 861602) and the University of Antwerp and Research Scientific Foundation Flanders (FWO, project number: G060920N) for the funding of their research. The Slovenian Research Agency (ARRS) Programme group P1–0143 and ARRS-ARRS Flanders Research Foundation (project: N1–0143) is also acknowledged. The Italian group thank the Dipartimento Politiche Antidroga (Presidenza del Consiglio dei Ministri, Rome; "Aque reflue" project) for partial funding of this research, and Metropolitana Milanese for sampling collection. Samples from Arizona were acquired with partial support from an award by the J.M. Kaplan Fund to RUH: OneWaterOneHealth nonprofit project 30009070 of the Arizona State University Foundation. The views expressed in this article are those of the author(s) and not necessarily those of the funding agencies.

Background Gastric non-Helicobacter pylori Helicobacter (NHPH) species naturally associated with animals have been linked with gastric disease in human patients.Aim The prevalence and clinical significance of zoonotic gastric NHPHs was determined in large and well-defined, H. pylori-negative, gastric patient populations.Methods Patients were retrospectively (n = 464) and prospectively (n = 65) included for gastric biopsy collection: chronic gastritis (CG), peptic ulcer disease and gastric MALT lymphoma, without identified aetiology. PCR and sequencing was performed for the detection of gastric Helicobacter species. Retrospectively, asymptomatic gastric bypass patients (n = 38) were included as controls. Prospectively, additional saliva samples and symptom and risk factor questionnaires were collected. In this group, patients with gastric NHPH infection were administered standard H. pylori eradication therapy and underwent follow-up gastroscopy post-therapy.Results In the retrospective samples, the prevalence of gastric NHPHs was 29.1%, while no gastric NHPHs were detected in control biopsies. In the prospective cohort, a similar proportion tested positive: 27.7% in gastric tissue and 20.6% in saliva. The sensitivity and accuracy for the detection of gastric NHPHs in saliva compared to gastric tissue was 27.8% and 69.8% respectively. Following eradication therapy, clinical remission was registered in 12 of 17 patients, histological remission in seven of nine and eradication in four of eight patients.Conclusion These findings suggest a pathophysiological involvement of NHPHs in gastric disease. Patients presenting with gastric complaints may benefit from routine PCR testing for zoonotic gastric NHPHs.

A therapeutic combination of azithromycin (AZM) and colistin methanesulfonate (CMS) was shown to be effective against both non-PDR and PDR Klebsiella pneumoniae biofilms in vitro. These anti-biofilm effects, however, may not correlate with effects observed in standard plate assays, nor will they representative of in vivo therapeutic action. After all, biofilm-associated infection processes are also impacted by the presence of wound bed components, such as host cells or wound fluids, which can all affect the antibiotic effectiveness. Therefore, an in vitro wound model of biofilm infection which partially mimics the complex microenvironment of infected wounds was developed to investigate the therapeutic potential of an AZM-CMS combination against XDR K. pneumoniae isolates. The model consists of a 3D collagen sponge-like scaffold seeded with HEK293 cells submerged in a fluid milieu mimicking the wound bed exudate. Media that were tested were all based on different strengths of Dulbecco’s modified Eagles/high glucose medium supplemented with fetal bovine serum, and/or Bacto Proteose peptone. Use of this model confirmed AZM to be a highly effective antibiofilm component, when applied alone or in combination with CMS, whereas CMS alone had little antibacterial effectiveness or even stimulated biofilm development. The wound model proposed here proves therefore, to be an effective aid in the study of drug combinations under realistic conditions.

Gonorrhoea is the second most prevalent bacterial sexually transmittable infection (STI) worldwide. The bacterium Neisseria gonorrhoeae has developed resistance to all currently and previously recommended antibiotics used to treat it. In the first section of this thesis, we investigate the factors underpinning resistance in STI core-groups. We characterized the molecular pathways via which azithromycin resistance emerges in N. gonorrhoeae. In addition to the known macrolide resistance conferring mutations at the target binding site and the efflux pump, we discovered novel ribosomal protein mutations. We show that a population exposed to high levels of antibiotics due to frequent STI screening, has more resistant commensal Neisseria species in their throat compared to the general population. As commensal Neisseria species play an important role in the evolution of antimicrobial resistance in the pathogenic N. gonorrhoeae, including them in monitoring activities could serve as an early warning system. In the second section of this thesis, we evaluate new antibiotic sparing treatment options for gonorrhoea. Although we were unable to isolate bacteriophages effective against currently circulating N. gonorrhoeae strains, throat samples tested did exhibit antigonococcal activity. Bacteriocins, which could be promising alternatives to antibiotics and offer a novel strategy to prevent and treat gonorrhoeae infection, may be responsible for the observed lysis. Next, we assessed the consequences of using mouthwashes as alternative treatment. We report that chlorhexidine sensitivity might diminish after prolonged exposure, and even more concerning, it may cause cross-resistance to antibiotics currently used to treat gonorrhoea. Resistance to Listerine could not be induced, but daily Listerine use did influence the oropharyngeal microbiome. These findings imply that mouthwash applications may have unintended consequences and should be carefully considered.

IntroductionProper recognition and individualized therapy of vulvovaginal atrophy (VVA) is paramount.Areas coveredAssessment of VVA should be performed using several questionnaires in combination with wet mount microscopy to determine Vaginal Cell Maturation Index (VCMI) and infections. PubMed searches were carried out between 1 march 2022 and 15 October 2022.Low dose vaginal estriol seems safe, efficient, and could be used in patients with contraindications for steroid hormones such as women with a history of breast cancer, and should therefore be considered as first choice hormonal treatment, when non-hormonal treatments fail. New estrogens, androgens, and several Selective Estrogen Receptor Modulators (SERMs) are being developed and tested. Intravaginal Hyaluronic Acid (HA) or Vit D can help women who can't or don't want to use hormones.Expert opinionProper treatment is not possible without a correct and full diagnosis, including microscopy of the vaginal fluid. Low dose vaginal estrogen treatment, especially with estriol, is very efficient and is preferred in most women with VVA. Oral ospemifene and vaginal dihydroepiandrosterone (DHEA) are now considered efficient and safe alternative therapies for VVA. More safety data are waited for several SERMs and for a newly introduced estrogen: estetrol (E4), although so far no major side effects were seen from these drugs. Indications for laser treatments are questionable.

Head and neck cancer is a broad family of diseases, most of which are of squamous cell origin, affecting the epithelial mucosa lining the upper aerodigestive tract. They often recur or are progressive despite multimodality treatment approaches, resulting in a poor prognosis. Given the progressive aging of the global population, the probability to plan an active and eventually toxic treatment for an older patient, with either curative or palliative intent, can no longer be considered as an uncommon occurrence. A crucial point in offering a systemic treatment to older patients with head and neck squamous cell carcinoma is that they are underrepresented in randomised clinical trials, and evidence-based guidelines are lacking, while, from a clinical point of view, these patients may have varying grades of resilience to anticancer treatments due to differences in their health, social and/or economic status. Our aim is to draw attention to the older patient population suffering from recurrent and/or metastatic head and neck squamous cell carcinoma and to address some open questions, such as possible differences in epidemiology and biology compared with their younger counterparts; to highlight frailty and its components by discussing how to measure and use it to personalise treatment; to evaluate which outcomes should be best achieved in the older adult setting; finally, in the era of immunotherapy, to examine whether there are differences to be addressed when considering new treatments for older patients.

Objectives: Despite distinct clinical profiles, amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) patients share a remarkable portion of pathological features, with a substantial percentage of patients displaying a mixed disease phenotype. Kynurenine metabolism seems to play a role in dementia-associated neuroinflammation and has been linked to both diseases. We aimed to explore dissimilarities in kynurenine pathway metabolites in these early onset neurodegenerative disorders in a brain-region-specific manner. Methods: Using liquid chromatography mass spectrometry (LC-MS/MS), kynurenine metabolite levels were determined in the brain samples of 98 healthy control subjects (n = 20) and patients with early onset Alzheimer’s disease (EOAD) (n = 23), ALS (n = 20), FTD (n = 24) or a mixed FTD–ALS (n = 11) disease profile. Results: Overall, the kynurenine pathway metabolite levels were significantly lower in patients with ALS compared to FTD, EOAD and control subjects in the frontal cortex, substantia nigra, hippocampus and neostriatum. Anthranilic acid levels and kynurenine-to-tryptophan ratios were consistently lower in all investigated brain regions in ALS compared to the other diagnostic groups. Conclusions: These results suggest that the contribution of kynurenine metabolism in neuroinflammation is lower in ALS than in FTD or EOAD and may also be traced back to differences in the age of onset between these disorders. Further research is necessary to confirm the potential of the kynurenine system as a therapeutic target in these early onset neurodegenerative disorders.