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Contains fulltext : 174845.pdf (Publisher’s version ) (Open Access) The combination of methotrexate with epidermal growth factor receptor (EGFR) recombinant antibody, cetuximab, is currently being investigated in treatment of head and neck carcinoma. As methotrexate is cleared by renal excretion, we studied the effect of cetuximab on renal methotrexate handling. We used human conditionally immortalized proximal tubule epithelial cells overexpressing either organic anion transporter 1 or 3 (ciPTEC-OAT1/ciPTEC-OAT3) to examine OAT1 and OAT3, and the efflux pumps breast cancer resistance protein (BCRP), multidrug resistance protein 4 (MRP4), and P-glycoprotein (P-gp) in methotrexate handling upon EGF or cetuximab treatment. Protein kinase microarrays and knowledge-based pathway analysis were used to predict EGFR-mediated transporter regulation. Cytotoxic effects of methotrexate were evaluated using the dimethylthiazol bromide (MTT) viability assay. Methotrexate inhibited OAT-mediated fluorescein uptake and decreased efflux of Hoechst33342 and glutathione-methylfluorescein (GS-MF), which suggested involvement of OAT1/3, BCRP, and MRP4 in transepithelial transport, respectively. Cetuximab reversed the EGF-increased expression of OAT1 and BCRP as well as their membrane expressions and transport activities, while MRP4 and P-gp were increased. Pathway analysis predicted cetuximab-induced modulation of PKC and PI3K pathways downstream EGFR/ERBB2/PLCg. Pharmacological inhibition of ERK decreased expression of OAT1 and BCRP, while P-gp and MRP4 were increased. AKT inhibition reduced all transporters. Exposure to methotrexate for 24 h led to a decreased viability, an effect that was reversed by cetuximab. In conclusion, cetuximab downregulates OAT1 and BCRP while upregulating P-gp and MRP4 through an EGFR-mediated regulation of PI3K-AKT and MAPKK-ERK pathways. Consequently, cetuximab attenuates methotrexate-induced cytotoxicity, which opens possibilities for further research into nephroprotective comedication therapies.

Contains fulltext : 169651.pdf (Publisher’s version ) (Closed access) Sepsis-associated acute kidney injury is a multifactorial syndrome in which inflammation and renal microcirculatory dysfunction play a profound role. Subsequently, renal tubule mitochondria reprioritize cellular functions to prevent further damage. Here, we investigated the putative protective effects of human recombinant alkaline phosphatase (recAP) during inhibition of mitochondrial respiration in conditionally immortalized human proximal tubule epithelial cells (ciPTEC). Full inhibition of mitochondrial oxygen consumption was obtained after 24h antimycin A treatment, which did not affect cell viability. While recAP did not affect the antimycin A-induced decreased oxygen consumption and increased hypoxia-inducible factor-1alpha or adrenomedullin gene expression levels, the antimycin A-induced increase of pro-inflammatory cytokines IL-6 and IL-8 was attenuated. Antimycin A tended to induce the release of detrimental purines ATP and ADP, which reached statistical significance when antimycin A was co-incubated with lipopolysaccharide, and were completely converted into cytoprotective adenosine by recAP. As the adenosine A2A receptor was up-regulated after antimycin A exposure, an adenosine A2A receptor knockout ciPTEC cell line was generated in which recAP still provided protection. Together, recAP did not affect oxygen consumption but attenuated the inflammatory response during impaired mitochondrial function, an effect suggested to be mediated by dephosphorylating ATP and ADP into adenosine.

In this review we provide an overview of the expanding molecular toolbox that is available for gene based therapies and how these therapies can be used for a large variety of kidney diseases. Gene based therapies range from restoring gene function in genetic kidney diseases to steering complex molecular pathways in chronic kidney disorders, and can provide a treatment or cure for diseases that otherwise may not be targeted. This approach involves the delivery of recombinant DNA sequences harboring therapeutic genes to improve cell function and thereby promote kidney regeneration. Depending on the therapy, the recombinant DNA will express a gene that directly plays a role in the function of the cell (gene addition), that regulates the expression of an endogenous gene (gene regulation), or that even changes the DNA sequence of endogenous genes (gene editing). Some interventions involve permanent changes in the genome whereas others are only temporary and leave no trace. Efficient and safe delivery are important steps for all gene based therapies and also depend on the mode of action of the therapeutic gene. Here we provide examples on how the different methods can be used to treat various diseases, which technologies are now emerging (such as gene repair through CRISPR/Cas9) and what the opportunities, perspectives, potential and the limitations of these therapies are for the treatment of kidney diseases.

Contains fulltext : 172570.pdf (Publisher’s version ) (Closed access) Apical transport is key in renal function, and the activity of efflux transporters and receptor-mediated endocytosis is pivotal in this process. The conditionally immortalized proximal tubule epithelial cell line (ciPTEC) endogenously expresses these systems. Here, we used ciPTEC to investigate the activity of three major efflux transporters, viz., breast cancer resistance protein (BCRP), multidrug resistance protein 4 (MRP4), and P-glycoprotein (P-gp), as well as protein uptake through receptor-mediated endocytosis, using a fluorescence-based setup for transport assays. To this end, cells were exposed to Hoechst33342, chloromethylfluorescein-diacetate (CMFDA), and calcein-AM in the presence or absence of model inhibitors for BCRP (KO143), P-gp (PSC833), or MRPs (MK571). Overexpression cell lines MDCKII-BCRP and MDCKII-P-gp were used as positive controls, and membrane vesicles overexpressing one transporter were used to determine substrate and inhibitor specificities. Receptor-mediated endocytosis was investigated by determining the intracellular accumulation of fluorescently labeled receptor-associated protein (RAP-GST). In ciPTEC, BCRP and P-gp showed similar expressions and activities, whereas MRP4 was more abundantly expressed. Hoechst33342, GS-MF, and calcein are retained in the presence of KO143, MK571, and PSC833, showing clearly redundancy between the transporters. Noteworthy is the fact that both KO143 and MK571 can block BCRP, P-gp, and MRPs, whereas PSC833 appears to be a potent inhibitor for BCRP and P-gp but not the MRPs. Furthermore, ciPTEC accumulates RAP-GST in intracellular vesicles in a dose- and time-dependent manner, which was reduced in megalin-deficient cells. In conclusion, fluorescent-probe-based assays are fast and reproducible in determining apical transport mechanisms, in vitro. We demonstrate that typical substrates and inhibitors are not specific for the designated transporters, reflecting the complex interactions that can take place in vivo. The set of tools we describe are also compatible with innovative kidney culture models and allows studying transport mechanisms that are central to drug absorption, disposition, and detoxification.

Item does not contain fulltext The relevance of membrane transporters gained momentum in recent years and it is now widely recognized that transporters are key players in drug disposition and chemoresistance. As such, the kidneys harbor a variety of drug transporters and are one of the main routes for xenobiotic excretion. The breast cancer resistance protein (BCRP/ABCG2) is widely accepted as a key mediator of anticancer drug resistance and is a prominent renal drug transporter. Here, we review the role of BCRP in both processes and present a multitude of variables that can influence its activity. An increasing number of renally cleared chemotherapeutics, including tyrosine kinase inhibitors, described as BCRP substrates can modulate its activity via transcription factors and cellular signaling pathways, such as the phosphoinositide 3-kinase (PI3K) pathway. In addition to pharmacological actions, genetic variations, as well as differences between species and gender can affect BCRP function, which are also discussed. Furthermore, the role of BCRP in light of cancer treatments and the implications for novel therapeutic interventions that take into account renal function are discussed.

Contains fulltext : 172030.pdf (Publisher’s version ) (Open Access) The development of a biotechnological platform for the removal of waste products (e.g. uremic toxins), often bound to proteins in plasma, is a prerequisite to improve current treatment modalities for patients suffering from end stage renal disease (ESRD). Here, we present a newly designed bioengineered renal tubule capable of active uremic toxin secretion through the concerted action of essential renal transporters, viz. organic anion transporter-1 (OAT1), breast cancer resistance protein (BCRP) and multidrug resistance protein-4 (MRP4). Three-dimensional cell monolayer formation of human conditionally immortalized proximal tubule epithelial cells (ciPTEC) on biofunctionalized hollow fibers with maintained barrier function was demonstrated. Using a tailor made flow system, the secretory clearance of human serum albumin-bound uremic toxins, indoxyl sulfate and kynurenic acid, as well as albumin reabsorption across the renal tubule was confirmed. These functional bioengineered renal tubules are promising entities in renal replacement therapies and regenerative medicine, as well as in drug development programs.

Drug-induced nephrotoxicity still hampers drug development, because current translation from in vitro or animal studies to human lacks high predictivity. Often, renal adverse effects are recognized only during clinical stages of drug development. The current study aimed to establish a robust and a more complete human cell model suitable for screening of drug-related interactions and nephrotoxicity. In addition to endogenously expressed renal organic cation transporters and efflux transporters, conditionally immortalized proximal tubule epithelial cells (ciPTEC) were completed by transduction of cells with the organic anion transporter (OAT) 1 or OAT3. Fluorescence-activated cell sorting upon exposure to the OAT substrate fluorescein successfully enriched transduced cells. A panel of organic anions was screened for drug-interactions in ciPTEC-OAT1 and ciPTEC-OAT3. The cytotoxic response to the drug-interactions with antivirals was further examined by cell viability assays. Upon subcloning, concentration-dependent fluorescein uptake was found with a higher affinity for ciPTEC-OAT1 (Km = 0.8 ± 0.1 μM) than ciPTEC-OAT3 (Km = 3.7 ± 0.5 μM). Co-exposure to known OAT1 and/or OAT3 substrates (viz. para-aminohippurate, estrone sulfate, probenecid, furosemide, diclofenac, and cimetidine) in cultures spanning 29 passage numbers revealed relevant inhibitory potencies, confirming the robustness of our model for drug-drug interactions studies. Functional OAT1 was directly responsible for cytotoxicity of adefovir, cidofovir, and tenofovir, while a drug interaction with zidovudine was not associated with decreased cell viability. Our data demonstrate that human-derived ciPTEC-OAT1 and ciPTEC-OAT3 are promising platforms for highly predictive drug screening during early phases of drug development. Electronic supplementary material The online version of this article (doi:10.1208/s12248-016-9871-8) contains supplementary material, which is available to authorized users.

Contains fulltext : 170838.pdf (Publisher’s version ) (Open Access) BACKGROUND: To date, over 150 possible uremic solutes have been listed, but their role in the progression of CKD is largely unknown. Here, the association between a selected panel of uremic solutes and progression in CKD patients was investigated. METHODS: Patients from the MASTERPLAN study, a randomized controlled trial in CKD patients with a creatinine clearance between 20 and 70 ml/min per 1.73m2, were selected based on their rate of eGFR decline during the first five years of follow-up. They were categorized as rapid (decline >5 ml/min per year) or slow progressors. Concentrations of eleven uremic solutes were obtained at baseline and after one year of follow-up. Logistic regression was used to compare the odds for rapid to slow progression by uremic solute concentrations at baseline. Variability in uremic solute levels was assessed using scatter plots, and limits of variability were calculated. RESULTS: In total, 40 rapidly and 40 slowly progressing patients were included. Uremic solutes were elevated in all patients compared to reference values for healthy persons. The serum levels of uremic solutes were not associated with rapid progression. Moreover, we observed substantial variability in solute levels over time. CONCLUSIONS: Elevated concentrations of uremic solutes measured in this study did not explain differences in rate of eGFR decline in CKD patients, possibly due to lack of power as a result of the small sample size, substantial between patient variability, and variability in solute concentrations over time. The etiology of intra-individual variation in uremic solute levels remains to be elucidated.

Background: Hepcidin regulates systemic iron homeostasis by downregulating the iron exporter ferroportin. Circulating hepcidin is mainly derived from the liver but hepcidin is also produced in the heart. We studied the differential and local regulation of hepcidin gene expression in response to myocardial infarction (MI) and/or chronic kidney disease (CKD). We hypothesized that cardiac hepcidin gene expression is induced by and regulated to severity of cardiac injury, either through direct (MI) or remote (CKD) stimuli, as well as through increased local iron content. Methods: Nine weeks after subtotal nephrectomy (SNX) or sham surgery (CON), rats were subjected to coronary ligation (CL) or sham surgery to realize 4 groups: CON, SNX, CL and SNX + CL. In week 16, the gene expression of hepcidin, iron and damage markers in cardiac and liver tissues was assessed by quantitative polymerase chain reaction and ferritin protein expression was studied by immunohistochemistry. Results: Cardiac hepcidin messenger RNA (mRNA) expression was increased 2-fold in CL (p = 0.03) and 3-fold in SNX (p = 0.01). Cardiac ferritin staining was not different among groups. Cardiac hepcidin mRNA expression correlated with mRNA expression levels of brain natriuretic peptide (β = 0.734, p < 0.001) and connective tissue growth factor (β = 0.431, p = 0.02). In contrast, liver hepcidin expression was unaffected by SNX and CL alone, while it had decreased 50% in SNX + CL (p < 0.05). Hepatic ferritin immunostaining was not different among groups. Conclusions: Our data indicate differences in hepcidin regulation in liver and heart and suggest a role for injury rather than iron as the driving force for cardiac hepcidin expression in renocardiac failure.

Contains fulltext : 172437.pdf (Publisher’s version ) (Closed access) Two small clinical trials indicated that administration of bovine intestinal alkaline phosphatase (AP) improves renal function in critically ill patients with sepsis-associated acute kidney injury (AKI), for which the mechanism of action is not completely understood. Here, we investigated the effects of a newly developed human recombinant AP (recAP) on renal oxygenation and hemodynamics and prevention of kidney damage and inflammation in two in vivo AKI models. To induce AKI, male Wistar rats (n=18) were subjected to renal ischemia (30min) and reperfusion (I/R), or sham-operated. In a second model, rats (n=18) received a 30min infusion of lipopolysaccharide (LPS; 2.5mg/kg), or saline, and fluid resuscitation. In both models, recAP (1000U/kg) was administered intravenously (15min before reperfusion, or 90min after LPS). Following recAP treatment, I/R-induced changes in renal blood flow, renal vascular resistance and oxygen delivery at early, and cortical microvascular oxygen tension at late reperfusion were no longer significantly affected. RecAP did not influence I/R-induced effects on mean arterial pressure. During endotoxemia, recAP treatment did not modulate the LPS-induced changes in systemic hemodynamics and renal oxygenation. In both models, recAP did exert a clear renal protective anti-inflammatory effect, demonstrated by attenuated immunostaining of inflammatory, tubular injury and pro-apoptosis markers. Whether this renal protective effect is sufficient to improve outcome of patients suffering from sepsis-associated AKI is being investigated in a large clinical trial.

Item does not contain fulltext Evaluation of renal function is crucial for a number of clinical situations. Here, we reported a novel exogenous fluorescent marker (FITC-HPbetaCD) to real-time assess renal function by using a transcutaneous fluorescent detection technique. FITC-HPbetaCD was designed based on the principle of renal clearance of designed drugs. It displays favorable fluorescent properties, high hydrophilicity, low plasma protein binding, and high stability in porcine liver esterase as well as in plasma and nontoxicity. More importantly, FITC-HPbetaCD can be efficiently and rapidly filtered by glomerulus and completely excreted into urine without proximal tubular reabsorption or secretion in rat models. Additionally, the marker was well-tolerated, with nearly 100% urinary recovery of the given doses, and no metabolism were found. Relying on this novel kidney function marker and transcutaneous devices, we demonstrate a rapid, robust, and convenient approach for real-time assessing renal function without the need of time-consuming blood and urine sample preparation. Our work provides a promising tool for noninvasive real-time monitoring of renal function in vivo.

Objectives Alcohol and medication use are increasingly prevalent in the older population. Concurrent use of alcohol and alcohol-interactive (AI) medication can lead to significant adverse consequences. Methods Three reference works were used to create an explicit list of drug substances for which information about the interaction with alcohol was available in at least one of them. Additional information was extracted from the Summary of Product Characteristics (SPC). The first aim was to generate a list of 256 substances with standardized advice regarding the concurrent use of each drug with alcohol. The second aim was to observe the prevalence of potential drug–alcohol-interactions. The list was applied to a database containing information about alcohol and medication use of 1,016 community-dwelling older patients (≥70 years) with polypharmacy. Results About half of the sample population reported to consume alcohol at least once a week. Around 22% were classified as frequent drinkers (5–7 days/week) and 11% as heavier drinkers (>7 units/week). Ninety-three percent alcohol consumers in our sample took at least one chronic drug that potentially interacts with alcohol and 42% used at least one chronic drug for which alcohol use is considered contraindicated. Conclusions We developed an explicit list of potentially drug–alcohol-interactions in older adults, with standardized handling advice. We observed that prevalence of potential drug–alcohol-interactions is substantial in community-dwelling older patients with polypharmacy.

Background: Trauma-based psychotherapy is a first line treatment for post-traumatic stress disorder (PTSD) but not all patients achieve long-term remission. Transcranial direct current stimulation (tDCS) received considerable attention as a neuromodulation method that may improve trauma-based psychotherapy. Objective: We explored the effects of repeated anodal tDCS over the prefrontal cortex (PFC) on fear extinction in mice as a preclinical model for trauma-based psychotherapy. Methods: We performed auditory fear conditioning with moderate or high shock intensity on C57BL6/J mice. Next, mice received anodal tDCS (0.2 mA, 20 min) or sham stimulation over the PFC twice daily for five consecutive days. Extinction training was performed by repeatedly exposing mice to the auditory cue the day after the last stimulation session. Early and late retention of extinction were evaluated one day and three weeks after extinction training respectively. Results: We observed no significant effect of tDCS on the acquisition or retention of fear extinction in mice subjected to fear conditioning with moderate intensity. However, when the intensity of fear con-ditioning was high, tDCS significantly lowered freezing during the acquisition of extinction, regardless of the extinction protocol. Moreover, when tDCS was combined with a strong extinction protocol, we also observed a significant improvement of early extinction recall. Finally, we found that tDCS reduced generalized fear induced by contextual cues when the intensity of conditioning is high and extinction training limited. Conclusions: Our data provide a rationale to further explore anodal tDCS over the PFC as potential support for trauma-based psychotherapy for PTSD. (c) 2021 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Supplemental Digital Content is available in the text., Rationale: Endothelial cells (ECs) are highly glycolytic and generate the majority of their energy via the breakdown of glucose to lactate. At the same time, a main role of ECs is to allow the transport of glucose to the surrounding tissues. GLUT1 (glucose transporter isoform 1/Slc2a1) is highly expressed in ECs of the central nervous system (CNS) and is often implicated in blood-brain barrier (BBB) dysfunction, but whether and how GLUT1 controls EC metabolism and function is poorly understood. Objective: We evaluated the role of GLUT1 in endothelial metabolism and function during postnatal CNS development as well as at the adult BBB. Methods and Results: Inhibition of GLUT1 decreases EC glucose uptake and glycolysis, leading to energy depletion and the activation of the cellular energy sensor AMPK (AMP-activated protein kinase), and decreases EC proliferation without affecting migration. Deletion of GLUT1 from the developing postnatal retinal endothelium reduces retinal EC proliferation and lowers vascular outgrowth, without affecting the number of tip cells. In contrast, in the brain, we observed a lower number of tip cells in addition to reduced brain EC proliferation, indicating that within the CNS, organotypic differences in EC metabolism exist. Interestingly, when ECs become quiescent, endothelial glycolysis is repressed, and GLUT1 expression increases in a Notch-dependent fashion. GLUT1 deletion from quiescent adult ECs leads to severe seizures, accompanied by neuronal loss and CNS inflammation. Strikingly, this does not coincide with BBB leakiness, altered expression of genes crucial for BBB barrier functioning nor reduced vascular function. Instead, we found a selective activation of inflammatory and extracellular matrix related gene sets. Conclusions: GLUT1 is the main glucose transporter in ECs and becomes uncoupled from glycolysis during quiescence in a Notch-dependent manner. It is crucial for developmental CNS angiogenesis and adult CNS homeostasis but does not affect BBB barrier function.

The ghrelin system was previously proposed to mediate an independent branch of the stress response that curbs fear processing. Interestingly, the ghrelin system was also shown to control the activity of midbrain dopamine neurons. Given that dopamine neurons of the ventral tegmental area appear to have a critical role in fear processing, we aimed to investigate their contribution to the effects of ghrelin on fear processing. Our data show that systemic administration of the ghrelin receptor agonist MK0677, in a dose that induces food intake, has no significant effect on auditory fear processing and does not significantly affect dopamine release in the nucleus accumbens of male C57BL/6J mice. Local administration of the ghrelin receptor agonist MK0677 into the ventral tegmental area significantly increases food intake and it also significantly increased dopamine release in the nucleus accumbens, the medial prefrontal cortex and the amygdala. Nevertheless, it did not significantly affect auditory fear extinction. Our data indicate that pharmacological activation of midbrain dopamine neurons using a ghrelin receptor agonist does not affect auditory fear extinction. We also investigated the effect of non-pharmacological manipulation of the ghrelin system on auditory fear processing. However, we found that neither overnight food deprivation nor genetic ablation of the ghrelin receptor had a significant effect on auditory fear extinction. We conclude that the effects of manipulation of the ghrelin system on fear processing are subject to boundary conditions that remain poorly understood.

Introduction: Lemierre's syndrome is a septic thromboembolic complication of an oropharyngeal or neck infection, primarily caused by Fusobacterium species. Although it usually affects young healthy patients, some case reports describe this syndrome in older population.Methods: A case report and a systematic review of the literature were conducted to investigate the late onset of Lemierre's syndrome. Forty-one articles were selected for the qualitative analysis, 39 for the quantitative analysis.Results: The average age of the study population was 52 years old. Diabetes mellitus and upper gastro-intestinal malignancy, common comorbidities in the study population, might play a role in the development of late-onset Lemierre's syndrome. Empiric antibiotic treatment should cover Fusobacterium and Streptococcus species both, which may cooperate to induce purulent disease. Reported unfavourable outcome was more than expected.Conclusion: Lemierre's syndrome in adulthood may differ from the usual version. This disease may further pass unrecognized, if presented out of the expected age range. Nevertheless, early diagnosis and prompt treatment are a requisite to prevent morbidity and mortality, which may be higher in this older population.

Background Hypertrophic cardiomyopathy (HCM) related myocardial vascular remodelling may lead to the reduction of myocardial blood supply and a subsequent progressive loss of cardiac function. This process has been difficult to observe and thus their connection remains unclear. Here we used non-invasive myocardial blood flow sensitive CMR to show an impairment of resting myocardial perfusion in a mouse model of naturally occurring HCM. Methods We used a mouse model (DBA/2 J; D2 mouse strain) that spontaneously carries variants in the two most susceptible HCM genes—Mybpc3 and Myh7 and bears the key features of human HCM. The C57BL/6 J (B6) was used as a reference strain. Mice with either B6 or D2 backgrounds (male: n = 4, female: n = 4) underwent cine-CMR for functional assessment at 9.4 T. Left ventricular (LV) wall thickness was measured in end diastolic phase by cine-CMR. Quantitative myocardial perfusion maps (male: n = 5, female: n = 5 in each group) were acquired from arterial spin labelling (cine ASL-CMR) at rest. Myocardial perfusion values were measured by delineating different regions of interest based on the LV segmentation model in the mid ventricle of the LV myocardium. Directly after the CMR, the mouse hearts were removed for histological assessments to confirm the incidence of myocardial interstitial fibrosis (n = 8 in each group) and small vessel remodelling such as vessel density (n = 6 in each group) and perivascular fibrosis (n = 8 in each group). Results LV hypertrophy was more pronounced in D2 than in B6 mice (male: D2 LV wall thickness = 1.3 ± 0.1 mm vs B6 LV wall thickness = 1.0 ± 0.0 mm, p p global = 7.5 ± 0.6 vs B6 MBFglobal = 9.3 ± 1.6 ml/g/min, p global = 6.6 ± 0.8 vs B6 MBFglobal = 8.2 ± 2.6 ml/g/min, p p p p Conclusions Our work describes an imaging marker using cine ASL-CMR with a potential to monitor vascular and myocardial remodelling in HCM.

The utilization of serious games and simulations in health professional education has increased. The Pharmacy Game is one such concept that intersects gamification and simulation, in which pharmacy student teams competitively manage simulated pharmacies, a concept included in the pharmacy curricula of seven international universities. This study aimed to compare the implementation and conduct of the Pharmacy Game of participant universities and their students’ performance in the same educational task. Data were collected via a questionnaire completed by academic staff in April 2020, and the collation of results of the same patient case was conducted at each university (April 2020 to March 2021). The main results reflected differences in the game frequencies and the curricular approach (standalone or integrated course) and in the learning outcomes for the Pharmacy Game. Other differences were identified in the extent to which students of other professions were part of the game such as medical students or pharmacy assistants. Student case outcomes revealed similar strengths across the universities in patient communication and focus on safety, with variations identified as areas for improvement. Collation of the international utilization of the Pharmacy Game identified a broad spectrum of similar learning outcomes, inspiring a model of international core and aspirational learning outcomes. While the Pharmacy Game has been implemented with flexibility regarding the numbers of teams (4–10) and the duration of activity (12–36 days), all universities reported positive experiences and student outcomes, suggesting that the intervention represents a potential tool to deliver capstone learning experiences, promote interprofessional education, reinforce patient safety, and prepare pharmacy graduates for future practice.

Between 19 May and 12 June 2020, employees of the UZ Brussel were recruited in this study aiming to document the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) seroprevalence, to investigate the potential work-related risk factors for SARS-CoV-2 infection and to estimate the proportion of asymptomatic infections. In total, 2662 participants were included of whom 7.4% had immunoglobulin G antibodies against SARS-CoV-2. Of the participants reporting a positive polymerase chain reaction for SARS-CoV-2, 89% had antibodies at the time of blood sampling. Eleven per cent of the antibody positive participants reported no recent symptoms suggestive of coronavirus disease 2019 (COVID-19). Participants reporting fever, chest pain and/or anosmia/ageusia were significantly more frequently associated with the presence of antibodies against SARS-CoV-2. The presence of antibodies was highest in the group that had had contact with COVID-19-infected individuals outside the hospital with or without using appropriate personnel protective equipment (PPE) (P < 0.001). Inside the hospital, a statistically significant difference was observed for the employees considered as low-risk exposure compared to the intermediate-risk exposure group (P = 0.005) as well as the high-risk exposure group compared to the intermediate exposure risk group (P < 0.001). These findings highlight the importance of using correct PPE.

Objective Explicit screening tools and implicit evaluation methods have been developed to assist healthcare professionals in the management of pharmacotherapy in older adults. As prescribing habits and locally available medications vary considerably between countries, guides tailored to the needs of specific regions may be required. We aimed to report the results of the international Delphi validation study for the Turkish Inappropriate Medication use in the Elderly (TIME) criteria set, which aims to detect inappropriate prescribing in older adults in Eastern Europe. Methods The study was conducted between June 2019 and March 2020. Delphi rounds were conducted by the TIME international working group, which included 11 internationally recognized experts in geriatric pharmacotherapy as Delphi panelists. They were asked to indicate to what extent they agreed or disagreed with each TIME criterion, taking into account both the available evidence and their own experience. We used a five-point Likert scale from 1 (strongly agree) to 5 (strongly disagree) and an online software program (SurveyMonkey®) to grade the level of agreement. Criteria with a median value of 1 or 2 and a 75th centile value of 1 or 2 were accepted, and criteria with a median value > 2 were rejected. Those with a median value of 1 or 2 but a 75th centile value > 2 were retained, to be assessed in the following round. The initial list of Delphi criteria comprised 153 TIME items. Results After three Delphi rounds, 134 criteria were accepted and seven criteria were rejected, while 12 criteria did not achieve consensus, and so were not included in the final validated set of TIME criteria. Conclusion We developed the internationally validated TIME criteria set based on a Delphi process involving international experts. The validation study suggests that the TIME criteria set can be applied in both central and Eastern European settings. Further studies are needed to assess the utility and benefit of the TIME criteria in reducing inappropriate drug use and improving clinical outcomes. Supplementary Information The online version contains supplementary material available at 10.1007/s40266-021-00855-5.

Daratumumab, an anti-CD38 antibody, is effective in AL amyloidosis with low tumor burden. Data of daratumumab treatment in patients with AL amyloidosis but high tumor burden (≥10% bone marrow plasma cells) are limited. We report retrospective data of 10 consecutive patients with high tumor burden treated with daratumumab for relapsed/refractory AL amyloidosis. The median age at diagnosis was 62.3 years; all patients had cardiac involvement, and six (60%) patients had renal involvement. Median bone marrow plasma cell infiltration was 15% (range 10%-40%), and the median difference between involved and noninvolved free light-chains (dFLC) was 446 mg/L (range 102-1392 mg/L). Patients had a median of three prior lines of therapy, including bortezomib in all patients and lenalidomide in seven (70%) patients. The median time to first hematological response was 14 days (range 7-28 days), and the median time to best hematological response was 64 days (range 7-301 days). The hematological overall response was 90%, with high-quality response (≥ very good partial remission [VGPR]) in 70% of the patients. Fifty percent of the patients had a cardiac response after a median of 3.8 months (range 0.7-9.1). Infusion-related adverse events ≤ grade 2 occurred in seven (70%) patients and grade 3 adverse events in one patient. After a median follow-up time of 10 months, eight (80%) patients continued to receive daratumumab. We conclude that daratumumab is a very effective and safe treatment option in AL patients with relapsed/refractory disease and high disease burden at diagnosis. Daratumumab leads to rapid disease control and improvement of organ function.

Because Zika virus (ZIKV) can cause serious birth defects and is involved in cases of Guillain-Barre syndrome, the ZIKV outbreak in the American continent in 2015 resulted in an enormous need for ZIKV diagnostic tools. We evaluated the LIAISON® XL Zika Capture IgM test on 106 samples from patients, mainly travelers, with a confirmed or probable ZIKV infection. Sensitivity between 0 and 84 days after onset of symptoms was 92.5%. Specificity was evaluated on a panel of 56 samples known to cause possible cross-reactions. Cross-reaction with DENV antibodies was limited (10.5%) but false-positive results occurred in samples from patients with malaria, CMV and EBV infections.

Objective The cystine/glutamate antiporter system x c - could represent a new target for antiepileptogenic treatments due to its crucial roles in glutamate homeostasis and neuroinflammation. To demonstrate this, we compared epilepsy development and seizure susceptibility in xCT knockout mice (xCT-/- ) and in littermate controls (xCT+/+ ) in different chronic models of epilepsy. Methods Mice were surgically implanted with electrodes in the basolateral amygdala and chronically stimulated to develop self-sustained status epilepticus (SSSE); continuous video-electroencephalography monitoring was performed for 28 days after SE and hippocampal histopathology was assessed. Corneal kindling was induced by twice daily electrical stimulation at 6 Hz and maintenance of the fully kindled state was evaluated. Next, messenger RNA (mRNA) and protein levels of xCT and of the proteins involved in the phosphoinositide 3-kinase (PI3K)/Akt/glycogen synthase kinase 3β (GSK-3β)/eukaryotic initiation factor 2α (eIF2α)/activating transcription factor 4 (ATF4) signaling pathway were measured at different time points during epileptogenesis in NMRI mice treated with pilocarpine. Finally, the anticonvulsant effect of sulfasalazine (SAS), a nonselective system x c - inhibitor, was assessed against 6 Hz-evoked seizures in pilocarpine-treated mice. Results In the SSSE model, xCT-/- mice displayed a significant delayed epileptogenesis, a reduced number of spontaneous recurrent seizures, and less pronounced astrocytic and microglial activation. Moreover, xCT-/- mice showed reduced seizure severity during 6 Hz kindling development and a lower incidence of generalized seizures during the maintenance of the fully kindled state. In pilocarpine-treated mice, protein levels of the PI3K/Akt/GSK-3β/eIF2α/ATF4 pathway were increased during the chronic phase of the model, consistent with previous findings in the hippocampus of patients with epilepsy. Finally, repeated administration of SAS protected pilocarpine-treated mice against acute 6 Hz seizure induction, in contrast to sham controls, in which system x c - is not activated. Significance Inhibition of system x c - could be an attractive target for the development of new therapies with a potential for disease modification in epilepsy.

Purpose: Centrally authorised medicinal products (CAMPs) in the European Union may offer added therapeutic value (ATV) but may be linked to high prices and limited efficiency. Health technology assessment (HTA) and managed entry schemes (MES) may facilitate the reimbursement decision by providing reliable estimates of the medicinal product’s value and costs and by controlling the remaining uncertainty, respectively. We investigated the impact of HTA criteria and the initiation of a MES on the reimbursement decision of CAMPs in Belgium. Methods: We selected all reimbursement submissions for new centrally authorised medicinal products in the 2010–2015 period. We retrieved data relating to the reimbursement decision, the HTA outcome and the use of a managed entry scheme. Results: The decision of the Minister was available for 115 dossiers, covering 36 (31.3%) orphan medicinal products (OMPs) and 79 ATV products. A MES was used in 41 submissions. A positive reimbursement decision was obtained in 65% of cases. The significant factors affecting the reimbursement decision were the approval of ATV, the medical need if it was considered ‘important or major’ and the use of a managed entry scheme. Price, budget impact and efficiency had no significant impact. Conclusions: Added therapeutic value and high medical need increase the odds for a positive reimbursement decision. No impact could be demonstrated of the cost-related HTA criteria. Cost elements may be biased by the use of a confidential MES. Without a MES, only 53% of the centrally authorised medicinal products, including OMPs, are reimbursed in Belgium., SCOPUS: ar.j, info:eu-repo/semantics/published

Background: A high Neutrophil-to-Lymphocyte ratio (NLR) in patients with acute ischemic stroke (AIS) has been associated with post-stroke infections, but it's role as an early predictive biomarker for post-stroke pneumonia (PSP) and urinary tract infection (UTI) is not clear. Aim: To investigate the usefulness of NLR obtained within 24 h after AIS for predicting PSP and UTI in the first week. Methods: Clinical and laboratory data were retrieved from the University Hospital Brussels stroke database/electronic record system. Patients were divided into those who developed PSP or UTI within the first week after stroke onset and those who didn't. Receiver operating characteristics (ROC) curves and logistic regression analysis were used to identify independent predictors. Results: Five hundred and fourteen patients were included, of which 15.4% (n = 79) developed PSP and 22% (n = 115) UTI. In univariate analysis, NLR was significantly higher in patients who developed PSP (4.1 vs. 2.8, p < 0.001) but not in those who developed UTI (3.3 vs. 2.9, p = 0.074). Multiple logistic regression analysis for PSP showed that NLR, male gender, dysphagia, and stroke severity measured by the National Institutes of Health Stroke Scale (NIHSS), were independent predictors of PSP. For NLR alone, the area under the curve (AUC) in the ROC curve was 0.66 (95% CI = 0.59–0.73). When combining NLR ≥ 4.7 with age >75 years, male gender, NIHSS > 7, and dysphagia, the AUC increased to 0.84 (95% CI = 0.79–0.89). Conclusion: The NLR within 24 h after AIS appears to have no predictive value for post-stroke UTI, and is only a weak predictor for identifying patients at high risk for PSP. Its predictive value for PSP appears to be much stronger when incorporated in a prediction model including age, gender, NIHSS score, and dysphagia., SCOPUS: ar.j, info:eu-repo/semantics/published

Two colorimetric broth microdilution antifungal susceptibility tests were compared, Sensititre YeastOne and MICRONAUT-AM for nine antifungal agents. One hundred clinical Candida isolates were tested, representing a realistic population for susceptibility testing in daily practice. The reproducibility characteristics were comparable. Only for fluconazole, caspofungin, 5-flucytosine and amphotericin B, an essential agreement of ≥90% could be demonstrated. Sensititre minimal inhibitory concentrations (MICs) were systematically higher than MICRONAUT MICs for all antifungals, except for itraconazole. CLSI clinical breakpoints (CBPs) and epidemiological cut-off values (ECVs) were used for Sensititre MICs while for MICRONAUT the EUCAST CBPs and ECVs were used. Only fluconazole, micafungin, and amphotericin B had a categorical agreement of ≥90%. For fluconazole, micafungin, and amphotericin B the susceptibility proportions were comparable. Susceptibility proportion of posaconazole and voriconazole was higher using the MICRONAUT system. For itraconazole and anidulafungin, the susceptibility proportion was higher using Sensititre. It was not possible to determine the true MIC values or the correctness of a S/I/R result since both commercial systems were validated against a different reference method. These findings show that there is a significant variability in susceptibility pattern and consequently on use of antifungals in daily practice, depending on the choice of commercial system. ispartof: JOURNAL OF FUNGI vol:7 issue:5 ispartof: location:Switzerland status: published

In our tertiary care center, the reported susceptibility of E. coli blood isolates to amoxicillin/clavulanic acid exceeded 90% in 2005 and showed a progressive decrease to 50% by 2017. In this study, we investigate whether there is a real increase in resistant E. coli strains or if this apparent decline in reported susceptibility might be attributed to the substitution of CLSI by EUCAST guidelines in 2014. We randomly selected 237 E. coli blood isolates (stored at - 80 °C) from 1985 to 2018 and reassessed their MIC values, applying both the CLSI (fixed ratio of clavulanic acid) and EUCAST guidelines (fixed concentration of clavulanic acid). In parallel, the susceptibility of these isolates was retested by disk diffusion, according to the EUCAST guidelines. Whole genome sequencing was successfully performed on 233 of the 237 isolates. In only 130 of the 237 isolates (55.0%), testing according to the EUCAST and CLSI criteria delivered identical MIC values for amoxicillin/clavulanic acid. In 64 of the 237 isolates (27.0%), the MIC values diverged one dilution; in 38 (16.0%), two dilutions; and in five (2.1%), three dilutions. From these 107 discrepant results, testing according to EUCAST methodology revealed more resistant profiles in 93 E. coli strains (94.1%). Also, phenotypical susceptibility testing according to EUCAST guidelines tends to correlate better with the presence of beta-lactamase genes compared to CLSI testing procedure. This study highlights the low agreement between EUCAST and CLSI methodologies when performing MIC testing of amoxicillin/clavulanic acid. More strains are categorized as resistant when EUCAST guidelines are applied. The low agreement between EUCAST and CLSI was confirmed by WGS, since most of EUCAST resistant/CLSI sensitive isolates harbored beta-lactamase genes.

Background The search for new Alzheimer’s disease (AD) cerebrospinal fluid (CSF) and blood biomarkers with potential pathophysiological and clinical relevance continues, as new biomarkers might lead to improved early and differential diagnosis, monitoring of disease progression and might even identify new druggable targets. Melatonin might be an interesting biomarker as an inverse correlation between CSF melatonin levels, and severity of the neuropathology as measured by Braak stages has been described. Melatonin can be measured in different body fluids, such as CSF, blood, saliva and urine. Objectives The aim of this systematic review was to review all available studies regarding melatonin levels in different body fluids in the AD continuum and give an extensive overview of reported outcomes. Methods We included papers comparing melatonin levels between healthy controls and human patients belonging to the AD continuum. A systematic search of PubMed and Web of Science led to inclusion of 20 full-length English papers following exclusion of duplicates. Results This systematic literature search showed that disruptions in melatonin levels occur with age, but also in AD when compared to age-matched controls. Night-time melatonin levels were found to be lower in CSF and blood of AD patients as compared to controls. Literature was not conclusive regarding alterations in blood daytime melatonin levels or regarding saliva melatonin in AD patients. Decreased total and night-time melatonin production has been described in urine of AD patients. Conclusion Our systematic review shows evidence for disruptions in (night-time) melatonin levels in AD as compared to age-matched controls. Although more studies are needed to understand the contribution of disruption of the melatonergic system to the pathophysiology of AD, the potential anti-AD effects that have been attributed to melatonin, renders research on this topic relevant for the discovery of potential future treatment effects of melatonin for AD. The use of melatonin as potential blood biomarker for disease progression should also be further investigated.

Background/aimsSARS-CoV-2 is highly contagious. More evidence concerning extrapulmonary transmission routes such as the eyes is urgently needed. Although the humoral immune response is important in the viral containment, the local response in tears has not yet been studied. The aim of our study was twofold: to assess the prevalence of both SARS-CoV-2 RNA and antibodies in tear fluid.MethodsIn a first series, nasopharyngeal sampling and tear sampling by Schirmer test strips were performed in 26 acutely ill patients with COVID-19 to assess the presence of SARS-CoV-2 RNA by reverse transcription PCR. In a second series, IgG and IgA responses to SARS-CoV-2 spike protein in serum and tear fluid of convalescent individuals (n=22) were compared with control individuals (n=15) by ELISA.ResultsSARS-CoV-2 RNA was detected in tears of 7/26 (26.9%) patients with COVID-19. None of them had ocular symptoms. Convalescent individuals displayed a significant higher ratio of IgG (pConclusionsOur results demonstrate the presence of SARS-CoV-2 RNA and a local IgG and IgA immune response in tear fluid. These data confirm the possibility of SARS-CoV-2 transmission through tear fluid and the importance of the eye as a first defence against SARS-CoV-2, indicating the potential of tears as a non-invasive surrogate for serum in monitoring the host immune response.

Plaque rupture followed by intracoronary thrombus formation is recognized as the most common pathophysiological mechanism in acute coronary syndromes (ACS). The second most common underlying substrate for ACS is plaque erosion whose hallmark is thrombus formation without cap disruption. Invasive and non-invasive methods have emerged as a promising tool for evaluation of plaque features that either predict or detect plaque erosion. Optical coherence tomography (OCT), high-definition intravascular ultrasound (IVUS), near-infrared spectroscopy (NIRS), and near-infrared autofluorescence (NIRF) have been used to study plaque erosion. The detection of plaque erosion in the clinical setting, mainly facilitated by OCT, has shed light upon the complex pathophysiology underlying ACS not related to plaque rupture. Coronary computed tomography angiography (CCTA), which is to date the most commonly used non-invasive technique for coronary plaque evaluation, may also have a role in the evaluation of patients predisposed to erosion. Also, computational models enabling quantification of endothelial shear stress may pave the way to new research in coronary plaque pathophysiology. This review focuses on the recent imaging techniques for the evaluation of plaque erosion including invasive and non-invasive assessment.

Encrusted uropathy is a rare subacute to chronic inflammatory disorder caused by infection with urease-producing bacteria, mainlyCorynebacterium urealyticum. The disorder is characterized by urothelial deposition of struvite and carbonated apatite, resulting in encrustations and ulceronecrotic inflammation of the urothelium and surrounding tissues. Most commonly, encrusted uropathy is encountered in patients with predisposing conditions. The disease remains underdiagnosed. High urinary pH and negative conventional urine cultures should raise suspicion of the diagnosis. Prognosis is dependent on timely diagnosis and treatment installment, which consists of urological removal of encrustations in combination with urinary acidification and long-term antibiotic therapy.

Background: Nocturnal cerebrospinal fluid (CSF) and blood melatonin levels are altered in Alzheimer’s disease (AD). However, literature remains inconclusive on daytime blood melatonin levels. A positive correlation between melatonin levels and Mini-Mental State Examination (MMSE) scores in AD subjects has been evidenced following cross-sectional analyses. Whereas a correlation between serum and spinal CSF melatonin has been shown in healthy volunteers, an equal investigation in AD patients still has to be undertaken. Objective: 1) To evaluate whether serum melatonin levels correlate with spinal CSF melatonin levels in AD. 2) To compare daytime CSF and serum melatonin levels between patients with AD dementia, mild cognitive impairment due to AD, and healthy controls, and to evaluate whether melatonin can affect cognitive decline in AD. Methods: Subjects with AD and healthy controls included in two existing cohorts, of whom a CSF and serum sample was available at the neurobiobank and had at least 6 months of neuropsychological follow-up, were included in the present study. Melatonin concentrations were measured with liquid chromatography-mass spectrometry. Results: Daytime serum melatonin levels correlated with spinal CSF melatonin levels in AD (r = 0.751, p

Metabolic-associated fatty liver disease (MAFLD) is a chronic liver disease that affects about a quarter of the world population. MAFLD encompasses different disease stadia ranging from isolated liver steatosis to non-alcoholic steatohepatitis (NASH), fibrosis, cirrhosis and hepatocellular carcinoma. Although MAFLD is considered as the hepatic manifestation of the metabolic syndrome, multiple concomitant disease-potentiating factors can accelerate disease progression. Among these risk factors are diet, lifestyle, genetic traits, intake of steatogenic drugs, male gender and particular infections. Although infections often outweigh the development of fatty liver disease, pre-existing MAFLD could be triggered to progress towards more severe disease stadia. These combined disease cases might be underreported because of the high prevalence of both MAFLD and infectious diseases that can promote or exacerbate fatty liver disease development. In this review, we portray the molecular and cellular mechanisms by which the most relevant viral, bacterial and parasitic infections influence the progression of fatty liver disease and steatohepatitis. We focus in particular on how infectious diseases, including coronavirus disease-19, hepatitis C, acquired immunodeficiency syndrome, peptic ulcer and periodontitis, exacerbate MAFLD. We specifically underscore the synergistic effects of these infections with other MAFLD-promoting factors.

Polycyclic aromatic hydrocarbons (PAH) are a complex group of organic compounds, consisting of at least three fused aromatic rings, which are formed during combustion of organic matter. While some PAHs have been reported to have carcinogenic and/or mutagenic properties, another possible negative health impact is their endocrine disrupting potential. Therefore, the aim of this study was to determine both the agonistic and antagonistic endocrine activity of 9 environmentally relevant PAHs using three different CALUX bioassays: The AhR-CALUX, The ERα-CALUX and PPARγ-CALUX. For the PPARγ-CALUX anthracene, fluoranthene, pyrene and fluorene showed weak agonistic activity, whilst benzo(a)pyrene (B(a)P) was the only one exhibiting weak antagonistic activity. For the AhR-CALUX, chrysene was the only PAH that showed relatively strong agonist activity (except for B(a)P which was used as a standard). Pyrene, anthracene and fluoranthene showed weak AhR agonist activity. In the ERα-CALUX bioassay, fluoranthene had agonistic activity whilst B(a)P exhibited both agonistic and antagonistic activity (lowering E2 activity by 30%). Phenanthrene and anthracene had weak ERα agonist activities. These results indicate that certain PAHs have multiple modes of action and can activate/inhibit multiple receptor signaling pathways known to play critical roles in mediating endocrine disruption.

Ghrelin is a peptide hormone released by specialized X/A cells in the stomach and activated by acylation. Following its secretion, it binds to ghrelin receptors in the periphery to regulate energy balance, but it also acts on the central nervous system where it induces a potent orexigenic effect. Several types of stressors have been shown to stimulate ghrelin release in rodents, including nutritional stressors like food deprivation, but also physical and psychological stressors such as foot shocks, social defeat, forced immobilization or chronic unpredictable mild stress. The mechanism through which these stressors drive ghrelin release from the stomach lining remains unknown and, to date, the resulting consequences of ghrelin release for stress coping remain poorly understood. Indeed, ghrelin has been proposed to act as a stress hormone that reduces fear, anxiety- and depression-like behaviors in rodents but some studies suggest that ghrelin may - in contrast - promote such behaviors. In this review, we aim to provide a comprehensive overview of the literature on the role of the ghrelin system in stress coping. We discuss whether ghrelin release is more than a byproduct of disrupted energy homeostasis following stress exposure. Furthermore, we explore the notion that ghrelin receptor signaling in the brain may have effects independent of circulating ghrelin and in what way this might influence stress coping in rodents. Finally, we examine how the ghrelin system could be utilized as a therapeutic avenue in stress-related psychiatric disorders (with a focus on anxiety- and trauma-related disorders), for example to develop novel biomarkers for a better diagnosis or new interventions to tackle relapse or treatment resistance in patients.

Here we describe the LifeTime Initiative, which aims to track, understand and target human cells during the onset and progression of complex diseases, and to analyse their response to therapy at single-cell resolution. This mission will be implemented through the development, integration and application of single-cell multi-omics and imaging, artificial intelligence and patient-derived experimental disease models during the progression from health to disease. The analysis of large molecular and clinical datasets will identify molecular mechanisms, create predictive computational models of disease progression, and reveal new drug targets and therapies. The timely detection and interception of disease embedded in an ethical and patient-centred vision will be achieved through interactions across academia, hospitals, patient associations, health data management systems and industry. The application of this strategy to key medical challenges in cancer, neurological and neuropsychiatric disorders, and infectious, chronic inflammatory and cardiovascular diseases at the single-cell level will usher in cell-based interceptive medicine in Europe over the next decade., The LifeTime initiative is an ambitious, multidisciplinary programme that aims to improve healthcare by tracking individual human cells during disease processes and responses to treatment in order to develop and implement cell-based interceptive medicine in Europe.

Insulin-regulated aminopeptidase (IRAP, cystinyl aminopeptidase, CysAP) and aminopeptidase M (alanyl aminopeptidase, AlaAP) are closely related enzymes involved in cognitive, metabolic, and cardiovascular functions. These functions may be modulated by the type of fat used in the diet. In order to analyze a possible coordinated response of both enzymes we determined simultaneously their activities in frontal cortex, liver, and plasma of adult male rats fed diets enriched with fats differing in their percentages of saturated, mono or polyunsaturated fatty acids such as sesame, sunflower, fish, olive, Iberian lard, and coconut. The systolic blood pressure, food intake, body and liver weight as well as glucose and total cholesterol levels in plasma were measured. The type of fat in the diet influences the enzymatic activities depending on the enzyme and its location. These results suggest cognitive improvement properties for diets with predominance of polyunsaturated fatty acids. Physiological parameters such as systolic blood pressure, food intake, and biochemical factors such as cholesterol and glucose in plasma were also modified depending on the type of diet, supporting beneficial properties for diets rich in mono and polyunsaturated fatty acids. Inter-tissue correlations between the analyzed parameters were also modified depending on the type of diet. If the type of fat used in the diet modifies the behavior and relationship between CysAP and AlaAP in and between frontal cortex, liver and plasma, the functions in which they are involved could also be modified.

International audience; Although thousands of solid tumors have been sequenced to date, a fundamental under-sampling bias isinherent in current methodologies. This is caused by a tissue sample input of fixed dimensions (e.g., 6 mmbiopsy), which becomes grossly under-powered as tumor volume scales. Here, we demonstrate representative sequencing (Rep-Seq) as a new method to achieve unbiased tumor tissue sampling. Rep-Seq uses fixed residual tumor material, which is homogenized and subjected to next-generation sequencing. Analysis of intratumor tumor mutation burden (TMB) variability shows a high level of misclassification using current single-biopsy methods, with 20% of lung and 52% of bladder tumors having at least one biopsy with high TMB butlow clonal TMB overall. Misclassification rates by contrast are reduced to 2% (lung) and 4% (bladder) when a more representative sampling methodology is used. Rep-Seq offers an improved sampling protocol for tumor profiling, with significant potential for improved clinical utility and more accurate deconvolution of clonal structure.

Memories remain dynamic after consolidation, and when reactivated, they can be rendered vulnerable to various pharmacological agents that disrupt the later expression of memory (i.e., amnesia). Such drug-induced post-reactivation amnesia has traditionally been studied in AAA experimental designs, where a memory is initially created for a stimulus A (be it a singular cue or a context) and later reactivated and tested through exposure to the exact same stimulus. Using a contextual fear conditioning procedure in rats and midazolam as amnestic agent, we recently demonstrated that drug-induced amnesia can also be obtained when memories are reactivated through exposure to a generalization stimulus (GS, context B) and later tested for that same generalization stimulus (ABB design). However, this amnestic intervention leaves fear expression intact when at test animals are instead presented with the original training stimulus (ABA design) or a novel generalization stimulus (ABC design). The underlying mechanisms of post-reactivation memory malleability and of MDZ-induced amnesia for a generalization context remain largely unknown. Here, we evaluated whether, like typical CS-mediated (or AAA) post-reactivation amnesia, GS-mediated (ABB) post-reactivation amnesia displays key features of a destabilization-based phenomenon. We first show that ABB post-reactivation amnesia is critically dependent on prediction error at the time of memory reactivation and provide evidence for its temporally graded nature. In line with the known role of GluN2B-NMDA receptor activation in memory destabilization, we further demonstrate that pre-reactivation administration of ifenprodil, a selective antagonist of GluN2B-NMDA receptors, prevents MDZ-induced ABB amnesia. In sum, our data reveal that ABB MDZ-induced post-reactivation amnesia exhibits the hallmark features of a destabilization-dependent phenomenon. Implication of our findings for a reconsolidation-based account of post-reactivation amnesia are discussed. ispartof: Progress In Neuro-Psychopharmacology & Biological Psychiatry vol:108 pages:1-14 ispartof: location:England status: published

Background: In the human central nervous system (CN), resting astrocytes do not visually show endothelin-1 (ET-1)-like immunoreactivity. In patients with multiple sclerosis (MS), an inflammatory disorder of the CNS, high levels of ET-1 are found in reactive astrocytes in demyelinated plaques. ET-1 may contribute to the pathology of MS by interrupting the blood-brain-barrier, enhancing inflammatory responses, excitotoxicity and reducing cerebral blood flow.Methods: We used the human astrocytoma cell line 1321N1 to investigate the role of inflammatory cytokines involved in MS lesions (IL-1β, TNF-α, IFN-γ, LPS, IL-10, TGF-β) on astrocytic ET-1 upregulation. Prucalopride, rolipram, fenofibrate, fluoxetine, simvastatin, daglutril, and resveratrol were investigated as potential candidate drugs to suppress cytokine-induced astrocytic ET-1 production. Effects on ET-1 production were measured using both ELISA and RT-qPCR.Results and Conclusions: ET-1 secretion by astrocytoma cells was only stimulated by the pro-inflammatory cytokines IL-1β and TNF-α. Fluoxetine, simvastatin, and resveratrol significantly inhibited this IL-1β- and TNF-α-induced ET-1 production. Simvastatin and resveratrol significantly reduced ET-1 mRNA levels, indicating an effect at the level of transcription. Fluoxetine significantly reduced endothelin converting enzyme-1 mRNA levels, suggesting and effect at the level of protein-processing. The required concentrations of simvastatin (>0.1 µM) and resveratrol (>10 µM) cannot be achieved in humans using pharmacologically accepted doses. Fluoxetine exerted a significant inhibitory effect on ET-1 secretion at a concentration of 5 µM, which is pharmacologically achievable in human brain, but the effect was modest (

A large number of computer-based prediction methods to determine the potential of chemicals to induce mutations at the gene level has been developed over the last decades. Conversely, only few such methods are currently available to predict potential structural and numerical chromosome aberrations. Even fewer of these are based on the preferred testing method for this endpoint, i.e. the micronucleus test. For the present work, in vivo micronucleus test results of 718 structurally diverse compounds were collected and applied for the construction of new models by means of the freely available SARpy in silico model building software. Multiple QSAR models were created using parameter variation and manual verification of (non-) alerting structures. To this extent, the original set of 718 compounds was split into a training (80 %) and a test (20 %) set. SARpy was applied on the training set to automatically extract sets of rules by generating and selecting substructures based on their prediction performance whereas the test set was used to evaluate model performance. Five different splits were made randomly, each of which had a similar balance between positive and negative substances compared to the full dataset. All generated models were characterised by an overall better performance than existing free and commercial models for the same endpoint, while demonstrating high coverage.

In vivo determination of brain mediators plays an important role in providing insight in how the brain functions. For this purpose, targeted metabolomics can be a very useful tool. Targeted metabolomics detects and measures certain known low-molecular-weight biomolecules involved in signaling pathways and biochemical processes in the central nervous system. Microdialysis is a powerful technique to sample brain mediators in the central nervous system. Several analytical techniques that can possibly be coupled to microdialysis are available. However, selection of an appropriate technique should be considered carefully, since sensitivity and specificity are critical when measuring these mediators in volume-restricted microdialysis samples. This review outlines some of the commonly applied sampling methods and analytical techniques and discusses some of the challenges encountered during the in vivo determination of central nervous system mediators.

International audience; Due to the essential roles of glutamate, detection and response to a large range of extracellular concentrations of this excitatory amino acid are necessary for the fine-tuning of brain functions. Metabotropic glutamate receptors (mGluRs) are implicated in shaping the activity of many synapses in the central nervous system. Among the eight mGluR subtypes, there is increasing interest in studying the mGlu3 receptor which has recently been linked to various diseases, including psychiatric disorders. This receptor displays striking functional properties, with a high and, often, full basal activity, making its study elusive in heterologous systems. Here, we demonstrate that Cl− ions exert strong positive allosteric modulation of glutamate on the mGlu3 receptor. We have also identified the molecular and structural determinants lying behind this allostery: a unique interactive “chloride-lock” network. Indeed, Cl− ions dramatically stabilize the glutamate-induced active state of the extracellular domain of the mGlu3 receptor. Thus, the mGlu3 receptors’ large basal activity does not correspond to a constitutive activity in absence of agonist. Instead, it results mostly from a Cl−mediated amplified response to low ambient glutamate concentrations, such as those measured in cell media. This strong interaction between glutamate and Cl− ions allows the mGlu3 receptor to sense and efficiently react to sub-micromolar concentrations of glutamate, making it the most sensitive member of mGluR family.

Aims To test specific mono‐agonists to the glucagon‐like peptide‐1 receptor (GLP‐1R), glucagon receptor (GCGR) and glucose‐dependent insulinotropic peptide receptor (GIPR), individually and in combination, in a mouse model of diet‐induced non‐alcoholic steatohepatitis (NASH) and fibrosis in order to decipher the contribution of their activities and potential additive effects to improving systemic and hepatic metabolism. Materials and methods We induced NASH by pre‐feeding C57BL/6J mice a diet rich in fat, fructose and cholesterol for 36 weeks. This was followed by 8 weeks of treatment with the receptor‐specific agonists 1‐GCG (20 mg/kg twice daily), 2‐GLP1 (3 mg/kg twice daily) or 3‐GIP (30 mg/kg twice daily), or the dual (1 + 2) or triple (1 + 2 + 3) combinations thereof. A dual GLP‐1R/GCGR agonistic peptide, 4‐dual‐GLP1/GCGR (30 mg/kg twice daily), and liraglutide (100 mg/kg twice daily) were included as references. Results Whereas low‐dose 1‐GCG or 3‐GIP alone did not influence body weight, liver lipids and histology, their combination with 2‐GLP1 provided additional weight loss, reduction in liver triglycerides and improvement in histological disease activity score. Notably, 4‐dual‐GLP‐1R/GCGR and the triple combination of selective mono‐agonists led to a significantly stronger reduction in the histological non‐alcoholic fatty liver disease activity score compared to high‐dose liraglutide, at the same extent of body weight loss. Conclusions GCGR and GIPR agonism provide additional, body weight‐independent improvements on top of GLP‐1R agonism in a murine model of manifest NASH with fibrosis.

Metabolomics is the comprehensive study of small-molecule metabolites. Obtaining a wide coverage of the metabolome is challenging because of the broad range of physicochemical properties of the small molecules. To study the compounds of interest spectroscopic (NMR), spectrometric (MS) and separation techniques (LC, GC, supercritical fluid chromatography, CE) are used. The choice for a given technique is influenced by the sample matrix, the concentration and properties of the metabolites, and the amount of sample. This review discusses the most commonly used analytical techniques for metabolomic studies, including their advantages, drawbacks and some applications.

Currently, there is mounting evidence that intermolecular receptor-receptor interactions may result in altered receptor recognition, pharmacology and signaling. Heterobivalent ligands have been proven useful as molecular probes for confirming and targeting heteromeric receptors. This report describes the design and synthesis of novel heterobivalent ligands for dopamine D2 -like receptors (D2 -likeR) and the μ-opioid receptor (μOR) and their evaluation using ligand binding and functional assays. Interestingly, we identified a potent bivalent ligand that contains a short 18-atom linker and combines good potency with high efficacy both in β-arrestin 2 recruitment for μOR and MAPK-P for D4 R. Furthermore, this compound was characterized by a biphasic competition binding curve for the D4 R-μOR heterodimer, indicative of a bivalent binding mode. As this compound possibly bridges the D4 R-μOR heterodimer, it could be used as a pharmacological tool to further investigate the interactions of D4 R and μOR.

Intensive care unit (ICU) patients are often extremely vulnerable to suffer an infection from airborne pathogens. Therefore, an environmental outbreak of Aspergillus fumigatus in an ICU should be handled in a concise and efficient way. In this short report, we describe an outbreak of A. fumigatus in the ICU of a Belgian secondary care hospital. Following initial detection of a humidity problem and airborne A. fumigatus, the department of infection prevention took urgent measures for damage control and resolution of the problem. As such a timely resolution has been provided, with assurance of a clean and healthy environment for the critical patients. Keywords: Aspergillus, Intensive care, Contamination

The purpose of this study was to test a newly developed decontamination and fluidization kit for processing respiratory specimens for the detection of mycobacteria: the Myco-TB procedure (developed by Copan (Brescia, Italy)). This technique was compared with the Zephiran decontamination method in use in our hospital. Respiratory specimens (n = 387: 130 endotracheal/bronchial aspirates, 172 bronchoalveolar lavages and 55 sputa) submitted to the University Hospital of Brussels between January 2016 and March 2017 were included. All samples were divided into two aliquots: one was subjected to the Myco-TB method and one to the Zephiran technique prior to culture. The sensitivities for culture for the Zephiran technique on solid media, the Myco-TB method on solid media and Myco-TB combined with the MGIT™ system were respectively 67%, 87% and 89%. The contamination rates were 22% with both the Zephiran and Myco-TB method on solid media and only 4% with the Myco-TB kit combined with the MGIT™ system. For direct microscopy, the sensitivities of the Zephiran method and the Myco-TB method were equal (40%) when the centrifugation time was 20 min. The Myco-TB decontamination method is easy and rapid to perform. It is more sensitive for culture as compared to the Zephiran method and gives lower contamination levels when combined with the MGIT™ technique. When increasing the centrifugation step to 20 min, the sensitivity of direct microscopy is equal to the Zephiran method.

Summary Background Hospital-acquired infections caused by VIM-encoded metallo-β-lactamase-positive Pseudomonas aeruginosa are a major problem in intensive care units (ICUs) worldwide. A previous study conducted in the UZ Brussel hospital revealed that sink drains of the ICU were a possible source of various multidrug-resistant pathogenic bacteria. Aim To investigate the presence and persistence of VIM P. aeruginosa in the sink drains of the four adult ICUs and their role in nosocomial infections, emphasizing sink-to-patient transmission. Methods Thirty-six sinks located in the ICUs of the UZ Brussel were sampled and screened for the presence of VIM P. aeruginosa in August and October 2019. Whole-genome sequencing (WGS) was performed on all positive sink drain isolates together with 61 isolates from patients who were retrospectively selected (ICU patients 2019–2020, N = 46; non-ICU patients 2019, N = 6). Findings Twenty sinks were found positive for P. aeruginosa at both sampling time-points. WGS revealed that the predominating environmental cluster belonged to sequence type ST111. Ten additional STs were identified. VIM-2 was detected among all ST17 (N = 2) and ST111 (N = 14) sink drain isolates. Based on whole-genome multi-locus sequence typing analysis of all genomes, 15 clusters of highly related isolates were identified, of which seven included both sink drain and clinical isolates. Conclusion Our findings confirm that sink drains are a possible source of VIM-2 P. aeruginosa, probably after being contaminated with clinical waste from patients. Patients could be exposed to VIM-2 P. aeruginosa dispersed in their environment because of colonized sink drains.

Matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry has emerged as a reliable technique to identify molds involved in human diseases, including dermatophytes, provided that exhaustive reference databases are available. This study assessed an online identification application based on original algorithms and an extensive in-house reference database comprising 11,851 spectra (938 fungal species and 246 fungal genera). Validation criteria were established using an initial panel of 422 molds, including dermatophytes, previously identified via DNA sequencing (126 species). The application was further assessed using a separate panel of 501 cultured clinical isolates (88 mold taxa including dermatophytes) derived from five hospital laboratories. A total of 438 (87.35%) isolates were correctly identified at the species level, while 26 (5.22%) were assigned to the correct genus but the wrong species and 37 (7.43%) were not identified, since the defined threshold of 20 was not reached. The use of the Bruker Daltonics database included in the MALDI Biotyper software resulted in a much higher rate of unidentified isolates (39.76 and 74.30% using the score thresholds 1.7 and 2.0, respectively). Moreover, the identification delay of the online application remained compatible with real-time online queries (0.15 s per spectrum), and the application was faster than identifications using the MALDI Biotyper software. This is the first study to assess an online identification system based on MALDI-TOF spectrum analysis. We have successfully applied this approach to identify molds, including dermatophytes, for which diversity is insufficiently represented in commercial databases. This free-access application is available to medical mycologists to improve fungal identification.