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Objectives: Recent studies report a fluctuating course of attention-deficit/ hyperactivity disorder (ADHD) across development characterized by intermittent periods of remission and recurrence. In the Multimodal Treatment of ADHD (MTA) study, we investigated fluctuating ADHD including clinical expression over time, childhood predictors, and between- and within-person associations with factors hypothesized as relevant to remission and recurrence., Methods: Children with DSM-5 ADHD, combined type (N = 483), participating in the MTA adult follow-up were assessed 9 times from baseline (mean age = 8.46) to 16-year follow-up (mean age = 25.12). The fluctuating subgroup (63.8% of sample) was compared to other MTA subgroups on variables of interest over time., Results: The fluctuating subgroup experienced multiple fluctuations over 16 years (mean = 3.58 , SD = 1.36) with a 6- to 7-symptom within-person difference between peaks and troughs. Remission periods typically first occurred in adolescence and were associated with higher environmental demands (both between- and within-person), particularly at younger ages. Compared to other groups, the fluctuating subgroup demonstrated moderate clinical severity. In contrast, the stable persistent group (10.8%) was specifically associated with early and lasting risk for mood disorders, substance use problems in adolescence/ young adulthood, low medication utilization, and poorer response to childhood treatment. Protective factors were detected in the recovery group (9.1%; very low parental psychopathology) and the partial remission group (15.6%; higher rates of comorbid anxiety)., Conclusions: In the absence of specific risk or protective factors, individuals with ADHD demonstrated meaningful within-individual fluctuations across development. Clinicians should communicate this expectation and monitor fluctuations to trigger as-needed return to care. During remission periods, individuals with ADHD successfully manage increased demands and responsibilities., Trial Registration: ClinicalTrials.gov identifier: NCT00000388., (© Copyright 2024 Physicians Postgraduate Press, Inc.)

Background/Objectives : Metastatic posterior uveal melanoma (PUM) is one of the deadliest types of melanomas. Though the median survival is short, some patients with metastatic disease live for a long time. In this study, we investigated whether the anatomical location of the metastatic lesions is associated with differences in survival. Methods : One hundred and seventy-eight patients with metastatic PUM with baseline whole-body imaging were retrospectively included. The patients were divided into three groups based on the anatomical location of metastases: (1) exclusive liver metastases (hepatic pattern), (2) both hepatic and extrahepatic metastatic lesions (hepatic-extrahepatic pattern), and (3) exclusive extrahepatic lesions (extrahepatic pattern). Survival was investigated using Kaplan-Meier plots, log-rank test, and the Cox proportional hazard model. Results : In total, 95 patients (53%) presented with hepatic pattern, 66 patients (37%) presented with hepatic-extrahepatic pattern, and 17 patients (10%) presented with extrahepatic pattern. Overall survival was significantly longer in patients with extrahepatic pattern (median 17.0 months) compared to those with hepatic pattern (median 11.0 months) and hepatic-extrahepatic pattern (median 7.0 months) ( p < 0.001, log-rank test). Multivariate Cox regression analysis showed increased hazard ratios (HR) for hepatic pattern (HR 2.37, 95% CI 1.08-5.17, p = 0.031) and hepatic-extrahepatic pattern (3.25, 95% CI 1.42-7.41, p = 0.005) compared to extrahepatic pattern. Most patients with hepatic (95%) and hepatic-extrahepatic patterns (82%) were diagnosed with metastases by liver ultrasonography screening, whereas 81% of patients with extrahepatic pattern developed symptoms that led to the diagnosis. Conclusions : Extrahepatic pattern was associated with prolonged survival in patients with metastatic PUM, despite there being a larger proportion of symptomatic patients. It is therefore important to consider the anatomical location of the metastatic lesions when stratifying patients into clinical trials.

Importance: Soluble thrombomodulin is a marker of endotheliopathy, and iloprost may improve endothelial function. In patients with septic shock, high plasma levels of soluble thrombomodulin (>10 ng/mL) have been associated with worse organ dysfunction and mortality., Objective: To assess the effects of treatment with iloprost vs placebo on the severity of organ failure in patients with septic shock and plasma levels of soluble thrombomodulin higher than 10 ng/mL., Design, Setting, and Participants: This investigator-initiated, adaptive, parallel group, stratified, double-blind randomized clinical trial was conducted between November 1, 2019, and July 5, 2022, at 6 hospitals in Denmark. The trial had a maximum sample size of 380, with an interim analysis for futility only at 200 patients with 90 days of follow-up. In total, 279 adults in the intensive care unit (ICU) with septic shock and endotheliopathy were included., Interventions: Patients were randomized 1:1 to masked intravenous infusion of iloprost, 1 ng/kg/min (n = 142), or placebo (n = 137) for 72 hours., Main Outcomes and Measures: The primary outcome was mean daily Sequential Organ Failure Assessment (SOFA) score in the ICU adjusted for trial site and baseline SOFA score for the per-protocol population. SOFA scores for each of the 5 organ systems ranged from 0 to 4, with higher scores indicating more severe dysfunction (maximum score, 20). The secondary outcomes included serious adverse reactions and serious adverse events at 7 days and mortality at 90 days., Results: Of 279 randomized patients, data from 278 were analyzed (median [IQR] age, 69 [58-77] years; 171 (62%) male), 142 in the iloprost group and 136 in the placebo group. The trial was stopped for futility at the planned interim analysis. The mean [IQR] daily SOFA score was 10.6 (6.4-14.8) in the iloprost group and 10.5 (5.9-15.5) in the placebo group (adjusted mean difference, 0.2 [95% CI, -0.8 to 1.2]; P = .70). Mortality at 90 days in the iloprost group was 57% (81 of 142) vs 51% (70 of 136) in the placebo group (adjusted relative risk, 1.12 [95% CI, 0.91-1.40]; P = .33). Serious adverse events occurred in 26 of 142 patients (18%) for the iloprost group vs 20 of 136 patients (15%) for the placebo group (adjusted relative risk, 1.25 [95% CI, 0.73-2.15]; P = .52). Only 1 serious adverse reaction was observed., Conclusions and Relevance: In this randomized clinical trial of adults in the ICU with septic shock and severe endotheliopathy, infusion of iloprost, 1 ng/kg/min, for 72 hours did not reduce mean daily SOFA scores compared with placebo. In a clinical context, administration of iloprost will be unlikely to improve outcome in these patients., Trial Registration: ClinicalTrials.gov Identifier: NCT04123444.

Background: Following the mass recall of valsartan products with nitrosamine impurities in July 2018, the number of patients exposed to these products, the duration of exposure, and the potential for cancer remains unknown. Therefore, we assessed the extent and duration of use of valsartan products with a nitrosamine impurity in the United States, Canada, and Denmark., Methods: We conducted a retrospective cohort study using administrative healthcare data from the US FDA Sentinel System, four Canadian provinces that contribute to the Canadian Network for Observational Drug Effect Studies (CNODES), and the Danish National Prescription Registry. Patients, 18 years and older between May 2012 and December 2020 with a valsartan dispensing were identified in each database. Patients were followed from the date of valsartan dispensing until discontinuation. We defined four valsartan exposure categories based on nitrosamine impurity status; recalled generic products with confirmed NDMA/NDEA levels (recalled-tested); recalled generic products that were not tested (recalled); non-recalled generic and non-recalled branded products. In Denmark, the recalled-tested category was not included due to absence of testing data. The proportion and duration of use of valsartan episodes stratified by nitrosamine-impurity status was calculated., Results: We identified 3.3 and 2.8 million (United States) and 51.3 and 229 thousand (Canada) recalled-tested and recalled valsartan exposures. In Denmark, where valsartan exposure was generally low, there were 10 747 recalled exposures. Immediately after the recall notices were issued, there was increased rates of switching to a non-valsartan ARB. The mean duration of use of the recalled-tested products was 167 (±223.1) and 146 (±255.8) days in the United States and Canada respectively. For the recalled products, mean cumulative duration of use was 178 (±249.6), 269 (±397.3) and 166 (±251.0) days in the United States, Canada, and Denmark, respectively., Conclusion: In this cohort study, despite widespread use of recalled generic valsartan between 2012 and 2018, the duration of use was relatively short and probably did not pose an elevated risk of nitrosamine-induced cancer. However, since products with nitrosamine impurity could have been on the market over a 6-year period, patients exposed to these products for longer durations could have a potentially different risk of cancer., (© 2024 John Wiley & Sons Ltd. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.)

The control of metabolic networks is incompletely understood, even for glycolysis in highly studied model organisms. Direct real-time observations of metabolic pathways can be achieved in cellular systems with 13 C NMR using dissolution Dynamic Nuclear Polarization (dDNP NMR). The method relies on a short-lived boost of NMR sensitivity using a redistribution of nuclear spin states to increase the alignment of the magnetic moments by more than four orders of magnitude. This temporary boost in sensitivity allows detection of metabolism with sub-second time resolution. Here, we hypothesized that dDNP NMR would be able to investigate molecular phenotypes that are not easily accessible with more conventional methods. The use of dDNP NMR allows real-time insight into carbohydrate metabolism in a Gram-positive bacterium ( Lactoccocus lactis ), and comparison to other bacterial, yeast and mammalian cells shows differences in the kinetic barriers of glycolysis across the kingdoms of life. Nevertheless, the accumulation of non-toxic precursors for biomass at kinetic barriers is found to be shared across the kingdoms of life. We further find that the visualization of glycolysis using dDNP NMR reveals kinetic characteristics in transgenic strains that are not evident when monitoring the overall glycolytic rate only. Finally, dDNP NMR reveals that resting Lactococcus lactis cells use the influx of carbohydrate substrate to produce acetoin rather than lactate during the start of glycolysis. This metabolic regime can be emulated using suitably designed substrate mixtures to enhance the formation of the C4 product acetoin more than 400-fold. Overall, we find that dDNP NMR provides analytical capabilities that may help to clarify the intertwined mechanistic determinants of metabolism and the optimal usage of biotechnologically important bacteria.

Retinoblastoma is the most common eye cancer in children. It is caused by pathogenic alterations of both alleles of the tumor suppressor gene RB1. In heritable retinoblastoma, a constitutional RB1 variant predisposes the cells to tumor formation, and loss of the other allele is a prerequisite for the development of retinoblastoma. Heritable retinoblastoma is inherited in an autosomal dominant manner; however, the majority of cases are the result of a de novo pathogenic RB1 variant. Penetrance is usually high (>90%), but with marked inter-familial variability. In some families, penetrance is incomplete and family members who develop tumors tend to remain unilaterally affected. Moreover, some families with low penetrance also show a parent-of-origin effect. We describe a patient with unilateral retinoblastoma caused by a previously unreported likely pathogenic RB1 variant (c.1199T>C) that disrupts a highly conserved amino acid residue within the A-box functional domain. Segregation analysis showed that the variant had unusually low penetrance as nine non-affected family members carried the same variant. We emphasize the use of genetic analysis on tumor DNA for classifying the RB1 variant, and underline the challenges in clinical management and counseling of families carrying the specific RB1 variant., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Background: Hereditary anemias are a group of genetic diseases prevalent worldwide and pose a significant health burden on patients and societies. The clinical phenotype of hereditary anemias varies from compensated hemolysis to life-threatening anemia. They can be roughly categorized into three broad categories: hemoglobinopathies, membranopathies, and enzymopathies. Traditional therapeutic approaches like blood transfusions, iron chelation, and splenectomy are witnessing a paradigm shift with the advent of targeted treatments. However, access to these treatments remains limited due to lacking or imprecise diagnoses. The primary objective of the study is to establish accurate diagnoses for patients with hereditary anemias, enabling optimal management. As a secondary objective, the study aims to enhance our diagnostic capabilities., Results: The DAHEAN study is a nationwide cohort study that collects advanced phenotypic and genotypic data from patients suspected of having hereditary anemias from all pediatric and hematological departments in Denmark. The study deliberates monthly by a multidisciplinary anemia board involving experts from across Denmark. So far, fifty-seven patients have been thoroughly evaluated, and several have been given diagnoses not before seen in Denmark., Conclusions: The DAHEAN study and infrastructure harness recent advancements in diagnostic tools to offer precise diagnoses and improved management strategies for patients with hereditary anemias., (© 2024. The Author(s).)

Aim: Periodontitis is an inflammatory disease driven by opportunistic bacteria including Porphyromonas gingivalis and Fusobacterium nucleatum, where T-cell and NKT-cell responses to these bacteria in patients with periodontitis grade B or C are not fully elucidated. The objective is to determine if exaggerated proinflammatory Th-cell responses to periodontitis-associated bacteria, but not commensal bacteria, is a characteristic of increased periodontitis grade., Methods: Mononuclear cells from patients with periodontitis grade C (n = 26) or grade B (n = 33) and healthy controls (HCs; n = 26) were stimulated with P. gingivalis, F. nucleatum or the commensal bacteria, Staphylococcus epidermidis and Cutibacterium acnes. Cytokine production by different T-cell populations and FOXP3-expression by regulatory T cells were assessed by flow cytometry., Results: Compared to HCs, grade C patients had decreased frequencies of interleukin (IL)-10-producing CD4+ T cells before stimulation (p = .02) and increased frequencies of IFN-y-producing CD4+ T cells after stimulation with P. gingivalis (p = .0019). Grade B patients had decreased frequencies of FOXP3+ CD4+ T cells before (p = .030) before and after stimulation with anti-CD2/anti-CD3/anti-CD28-loaded beads (p = .047), P. gingivalis (p = .013) and S. epidermidis (p = .018). Clinical attachment loss correlated with the frequencies of IFN-y-producing Th1 cells in P. gingivalis- and F. nucleatum-stimulated cultures in grade B patients (p = .023 and p = .048, respectively) and with the frequencies of Th17 cells in P. gingivalis-stimulated cultures (p = .0062) in grade C patients. Patients with periodontitis grade C or grade B showed lower frequencies of IL-10-producing NKT cells than HCs in unstimulated cultures (p = .0043 and p = .027 respectively)., Conclusions: Both periodontitis groups showed decreased frequencies of immunoregulatory T-cell and NKT cell subsets at baseline. Clinical attachment loss correlated with P. gingivalis-induced Th17-responses in grade C patients and with Th1-responses in grade B patients when cells were stimulated with P. gingivalis, supporting that dysregulated pro-inflammatory T-cell responses to periodontitis-associated bacteria contribute to the pathogenesis of periodontitis., (© 2024 The Author(s). Journal of Periodontal Research published by John Wiley & Sons Ltd.)

Background: Increasing evidence indicates that periodontitis contributes to systemic low-grade inflammation. Porphyromonas gingivalis is strongly associated with periodontitis, and antibodies against the bacterium may be used as a serological proxy to account for periodontal status, when studying diseases associated with periodontitis. The aim of the present study is to identify an easily accessible and reliable serological biomarker for determination of periodontal status and oral carriage of the bacterium., Methods: Saliva and serum samples were collected from periodontally healthy controls (n = 27), and patients with periodontitis stage II (n = 12) or stages III or IV (n = 44). Serum levels of immunoglobulin G (IgG) antibodies against intact and fragmented P. gingivalis, recombinant gingipains (RgpA and RgpB), and the bacteria Escherichia coli and Capnocytophaga ochracea as controls were quantified with a multiplex bead-based assay. P. gingivalis was identified in saliva using quantitative polymerase chain reaction (qPCR)., Results: Serum IgG antibodies against P. gingivalis whole bacteria were good indicators of periodontitis (area under the curve [AUC]: 0.75, 95% confidence interval [CI]: 0.64-0.85). The same was observed for levels of antibodies against P. gingivalis fragments (AUC: 0.78, 95% CI: 0.68-0.88). Likewise, levels of antibodies against P. gingivalis whole bacteria or P. gingivalis fragments were good indicators of oral carriage of P. gingivalis (AUC: 0.92, 95% CI: 0.86-0.98 and AUC: 0.96, 95% CI: 0.92-1, respectively). Conversely, antibodies against recombinant RgpA and RgpB were not good indicators of periodontitis or oral carriage of the bacterium. None of the antibody levels differed significantly between stage II and stage III or IV periodontitis., Conclusion: Serum IgG antibody levels against heat-inactivated whole P. gingivalis proved to be the preferable biomarker for periodontitis and oral carriage of the bacterium., (© 2024 The Authors. Journal of Periodontology published by Wiley Periodicals LLC on behalf of American Academy of Periodontology.)

Greenhouse cultivation has been expanding rapidly in recent years, yet little knowledge exists on its global extent and expansion. Using commercial and freely available satellite data combined with artificial intelligence techniques, we present a global assessment of greenhouse cultivation coverage and map 1.3 million hectares of greenhouse infrastructures in 2019, a much larger extent than previously estimated. Our analysis includes both large (61%) and small-scale (39%) greenhouse infrastructures. Examining the temporal development of the 65 largest clusters (>1,500 ha), we show a recent upsurge in greenhouse cultivation in the Global South since the 2000s, including a dramatic increase in China, accounting for 60% of the global coverage. We emphasize the potential of greenhouse infrastructures to enhance food security but raise awareness of the uncertain environmental and social implications that may arise from this expansion. We further highlight the gap in spatio-temporal datasets for supporting future research agendas on this critical topic., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

The aim of this study was to examine variations in use of antidepressants among children and adolescents in the three Scandinavian countries (Sweden, Norway, and Denmark). We identified new users of antidepressants (5-17 years) during 2007-2018 and described the annual incidence rate, treatment duration, concomitant psychotropic drug use, and the clinical setting of the prescribing physician (in Sweden and Denmark). Incident use of antidepressants increased by a factor 1.9 in Sweden, 1.3 in Norway and decreased by a factor 0.6 in Denmark during the study period. In Sweden, 58% of antidepressant users were covered by a prescription 12 months after initiation compared to 40% in Norway and 49% in Denmark. Also, 34% of Swedish antidepressant users were in continuous treatment after 12 months compared to 26% in Norway and 31% in Denmark. Concomitant use of other psychotropics was more common in Sweden (57%) than in Norway (37%) and Denmark (27%). During 2007-2018, clinicians from psychiatry settings initiated 75% of antidepressant treatments in Sweden, while this was the case for 50% of prescriptions in Denmark, although the proportion increased over time. The number of new antidepressant users is high and still rising in Sweden compared to Norway and Denmark. Swedish antidepressant users are more likely to use other psychotropics and to be covered by an antidepressant prescription after one year. Most antidepressants in Sweden are prescribed by physicians within psychiatric settings suggesting that they are based on specialized psychiatric evaluation., (© 2024. The Author(s).)

Purpose: This study aimed to systematically evaluate the validity of variables related to pregnancy, delivery, and key characteristics of the infant in the Danish National Patient Register using maternal medical records as the reference standard., Patients and Methods: We reviewed medical records of 1264 women giving birth in the Region of Southern Denmark during 2017. We calculated positive (PPV) and negative (NPV) predictive values, sensitivity, and specificity to estimate the validity of 49 selected variables., Results: The PPV was ≥0.90 on most pregnancy-related variables including parity, pre-gestational BMI, diabetes disorders, and previous cesarean section, while it was lower for hypertensive disorders, especially mild to moderate preeclampsia (0.49, 95% CI 0.32-0.66). Sensitivity ranged from 0.80 to 1.00 on all pregnancy-related variables, except hypertensive disorders (sensitivity 0.38-0.71, lowest for severe preeclampsia). On most delivery-related variables including obstetric surgical procedures (eg cesarean section and induction of labor), pharmacological pain-relief, and gestational age at delivery, PPV's ranged from 0.98 to 1.00 and the corresponding sensitivities from 0.87 to 1.00. Regarding infant-related variables, both the APGAR score registered five minutes after delivery and birthweight yielded a PPV of 1.00., Conclusion: Obstetric coding in the Danish National Patient Register shows very high validity and completeness making it a valuable source for epidemiologic research., Competing Interests: The authors report no financial or non-financial conflicts of interest in this work., (© 2024 Herskind et al.)

The demand for alternative sources of food proteins is increasing due to the limitations and challenges associated with conventional food production. Advances in biotechnology have enabled the production of proteins using microorganisms, thus prompting the exploration of attractive microbial hosts capable of producing functional proteins in high titers. Corynebacterium glutamicum is widely used in industry for the production of amino acids and has many advantages as a host organism for recombinant protein production. However, its performance in this area is limited by low yields of target proteins and high levels of native protein secretion. Despite representing a challenge for heterologous protein production, the C. glutamicum secretome has not been fully characterized. In this study, state-of-the-art mass spectrometry-based proteomics was used to identify and analyze the proteins secreted by C. glutamicum . Both the wild-type strain and a strain that produced and secreted a recombinant β-lactoglobulin protein were analyzed. A total of 427 proteins were identified in the culture supernatants, with 148 predicted to possess a secretion signal peptide. MS-based proteomics on the secretome enabled a comprehensive characterization and quantification (based on abundance) of the secreted proteins through label-free quantification (LFQ). The top 12 most abundant proteins accounted for almost 80% of the secretome. These are uncharacterized proteins of unknown function, resuscitation promoting factors, protein PS1, Porin B, ABC-type transporter protein and hypothetical membrane protein. The data can be leveraged for protein production by, e.g., utilizing the signal peptides of the most abundant proteins to improve secretion of heterologous proteins. In addition, secretory stress can potentially be alleviated by inactivating non-essential secreted proteins. Here we provide targets by identifying the most abundant, secreted proteins of which majority are of unknown function. The data from this study can thus provide valuable insight for researchers looking to improve protein secretion and optimize C. glutamicum as a host for secretory protein production., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Balasubramanian, Køhler, Jers, Jensen and Mijakovic.)

Short-term intake of selective serotonin reuptake inhibitors (SSRIs) modulates threat-related amygdala responses in healthy individuals. However, how SSRI intake over a clinically relevant time period modulates threat-related amygdala responses is less clear. In a semi-randomised, double-blind, placebo-controlled study of 64 healthy individuals (SSRI n = 32, placebo n = 32), we examined the effect of 3-5 weeks of SSRI escitalopram (20 mg daily) on brain response to angry, fearful and neutral faces using BOLD fMRI. Data was analysed using a whole-brain region-wise approach extracting standardised effects (i.e., Cohen's D). The study was conducted at the Copenhagen University Hospital. A priori, we hypothesised that SSRI would attenuate amygdala responses to angry and fearful faces but not to neutral ones. Whether SSRI modulates correlations between amygdala responses to emotional faces and negative mood states was also explored. Compared to placebo, 3-5 weeks of SSRI intake did not significantly affect the amygdala response to angry, fearful, or neutral faces (|Cohen's D|< 0.2, P FWER  = 1). Whole-brain, region-wise analyses revealed significant differences in frontal (|Cohen's D|< 0.6, P FWER  < .01) and occipital regions (|Cohen's D|< 0.5, P FWER  < .01). SSRI did not modulate correlations between amygdala responses to emotional faces and negative mood states. Our findings indicate that a 3-5 week SSRI intake impacts cortical responses to emotional stimuli, an effect possibly involved in SSRI's therapeutic efficacy.Trial registration Clinical Trials NCT04239339., (© 2024. The Author(s).)

Bacterial aerobic respiration may determine the outcome of antibiotic treatment in experimental settings, but the clinical relevance of bacterial aerobic respiration for the outcome of antibiotic treatment has not been tested. Therefore, we hypothesized that bacterial aerobic respiration is higher in sputum from patients with acute lower respiratory tract infections (aLRTI), than in sputum from patients with chronic LRTI (cLRTI), where the bacteria persist despite antibiotic treatment. The bacterial aerobic respiration was determined according to the dynamics of the oxygen (O 2 ) concentration in sputum from aLRTI patients (n = 52). This result was evaluated by comparison to previously published data from patients with cLRTI. O 2 consumption resulting in anoxic zones was more frequent in sputum with detected bacterial pathogens. The bacterial aerobic respiration in aLRTI sputum approximated 55% of the total O 2 consumption, which was significantly higher than previously published for cLRTI. The bacterial aerobic respiration in sputum was higher in aLRTI patients than previously seen in cLRTI patients, indicating the presence of bacteria with a sensitive physiology in aLRTI. These variations in bacterial physiology between aLRTI patients and cLRTI patients may contribute the huge difference in treatment success between the two patient groups., (© 2024 The Authors. APMIS published by John Wiley & Sons Ltd on behalf of Scandinavian Societies for Pathology, Medical Microbiology and Immunology.)

Purpose: To describe utilization patterns, characteristics of users and prescribers of the new oral antiviral medication, molnupiravir, indicated for mild-to-moderate COVID-19., Methods: Using nationwide registries, we identified all Danish adults who filled a prescription for molnupiravir from December 16th, 2021, to August 31st, 2022. We described weekly incidence rates and patient characteristics over time, prescriber characteristics as well as time between molnupiravir initiation and a positive SARs-CoV-2 test. Patient characteristics were compared to matched, untreated SARS-CoV-2 positive reference groups., Results: By August 31st, 2022, 5847 individuals had filled a prescription for molnupiravir. The incidence rate gradually increased to 16 weekly prescriptions per 1000 RT-PCR SARS-CoV-2 positives. Users of molnupiravir were most often men (55% vs. 45% women). The majority (81%) had a positive RT-PCR SARS-CoV-2 test and few (2.9%) redeemed molnupiravir outside the recommended window of 5 days from the positive test result. Compared to matched, untreated SARS-CoV-2 positive reference groups, users of molnupiravir had a median age of 74 years versus 49 years, a higher proportion resided in a nursing home (12% vs. 1.5%) and had a higher number of comorbidities (median of 3 vs. 0); most commonly hypertension (38%), chronic lung disease (35%), diabetes (20%) and mood disorders (20%). General practitioners were the primary prescribers of molnupiravir (91%)., Conclusions: Molnupiravir was mainly prescribed by general practitioners to RT-PCR SARS-CoV-2 positive individuals who had a potentially increased risk of severe COVID-19. Though some off-label prescribing occurred, our study indicates a high level of adherence to contemporary guidelines., (© 2023 The Authors. Pharmacoepidemiology and Drug Safety published by John Wiley & Sons Ltd.)

Objective: To compare the occurrence of sudden sensorineural hearing loss following immunization with BNT162b2 (Comirnaty®; Pfizer BioNTech) or mRNA-1273 (Spikevax®; Moderna) to the occurrence among unvaccinated individuals., Study Design: Cohort study., Setting: Nationwide Danish health care registers comprised all Danish residents living in Denmark on October 1, 2020, who were 18 years or older or turned 18 in 2021., Methods: We compared the occurrence of sudden sensorineural hearing loss following immunization with BNT162b2 (Comirnaty®; Pfizer BioNTech) or mRNA-1273 (Spikevax®; Moderna) (first, second, or third dose) against unvaccinated person time. Secondary outcomes were a first-ever hospital diagnosis of vestibular neuritis and a hearing examination, by an ear-nose-throat (ENT) specialist, followed by a prescription of moderate to high-dose prednisolone., Results: BNT162b2 or mRNA-1273 vaccine was not associated with an increased risk of receiving a discharge diagnosis of sudden sensorineural hearing loss (adjusted hazard ratio [HR]: 0.99, confidence interval [CI]: 0.59-1.64) or vestibular neuritis (adjusted HR: 0.94, CI: 0.69-1.24). We found a slightly increased risk (adjusted HR: 1.40, CI, 1.08-1.81) of initiating moderate to high-dose oral prednisolone following a visit to an ENT specialist within 21 days from receiving a messenger RNA (mRNA)-based Covid-19 vaccine., Conclusion: Our findings do not suggest an increased risk of sudden sensorineural hearing loss or vestibular neuritis following mRNA-based COVID-19 vaccination. mRNA-Covid-19 vaccination may be associated with a small excess risk of a visit to an ENT specialist visit followed by a prescription of moderate to high doses of prednisolone., (© 2023 The Authors. Otolaryngology-Head and Neck Surgery published by Wiley Periodicals LLC on behalf of American Academy of Otolaryngology-Head and Neck Surgery Foundation.)

This article presents a discussion inspired by the invitation formed by Kevin Carriere's book: "Psychology in Policy - Redefining Politics Through The Individual". From a theoretical standpoint in culture psychology Carriere challenges the idea of politics as a particular practice carried out by mainly politicians. Instead, he attempts to anchor processes of politics in the everyday lives of individuals, directed at changing their worlds. In this article, we discuss how this ambition could evolve even further by relating it to other theoretical approaches working with similar ambitions., (© 2023. The Author(s).)

Ocular tumours may arise from various tissues and therefore present as a heterogeneous group of diseases with unspecific symptoms. Some of the tumours carry a high mortality with a life expectancy less than 50% after ten years. Early diagnosis and treatment are essential for a good outcome, and centralization has led to a decreased morbidity and increased survival in Denmark. Tumour-specific somatic mutations can be used for personalized follow-up programmes and may lead to new treatment modalities, as argued in this review., (Published under Open Access CC-BY-NC-BD 4.0. https://creativecommons.org/licenses/by-nc-nd/4.0/.)

Background: Currently, bariatric surgery is the most effective long-term treatment of obesity. Sleeve gastrectomy (SG) and Roux-en-Y gastric bypass (RYGB) are the primary types of bariatric surgery performed worldwide. To minimize the risks of surgical complications and optimize cost-effectiveness, it is essential to develop fast-track protocols and patient logistics. At Aleris Hospitals in Denmark, a fast-track methodology in bariatric surgery has been implemented and continuously optimized over the last 15 years. The main objective was to demonstrate timelines recorded during one consecutive year in a fast-track, high-volume bariatric surgery setting after logistic optimization., Methods: This study included 949 consecutive patients who had undergone primary bariatric surgery in 2021. The primary outcomes were length of hospital stay and perioperative timeline recordings that were prospectively collected. The secondary outcomes were mortality, complication rates, and weight loss data., Results: The vast majority of our patients (99.1%) were discharged from the hospital within the day after surgery. The median total surgery time was 30 min, after 12 min of patient preparation and with a turnover time between patients of seven min. The median knife-to-knife time in one operating room was 56 min. Mortality was zero, 30-day reoperation rate was 1.2%, and 30-day readmission rate was 0.8%. SG and RYGB patients had an excess weight loss after four months of 45.6% and 57.9%, respectively., Conclusion: Implementation of fast-track principles in the clinical practice of bariatric surgery allows for an optimized, cost-effective surgical organization supporting the quality of procedures and patient safety., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The serotonin transporter (5-HTT) critically shapes serotonin neurotransmission by regulating extracellular brain serotonin levels; it remains unclear to what extent 5-HTT levels in the human brain are genetically determined. Here we applied [ 11 C]DASB positron emission tomography to image brain 5-HTT levels and evaluated associations with five common serotonin-related genetic variants that might indirectly regulate 5-HTT levels (BDNF rs6265, SLC6A4 5-HTTLPR, HTR1A rs6295, HTR2A rs7333412, and MAOA rs1137070) in 140 healthy volunteers. In addition, we explored whether these variants could predict in vivo 5-HTT levels using a five-fold cross-validation random forest framework. MAOA rs1137070 T-carriers showed significantly higher brain 5-HTT levels compared to C-homozygotes (2-11% across caudate, putamen, midbrain, thalamus, hippocampus, amygdala and neocortex). We did not observe significant associations for the HTR1A rs6295 and HTR2A rs7333412 genotypes. Our previously observed lower subcortical 5-HTT availability for rs6265 met-carriers remained in the presence of these additional variants. Despite this significant association, our prediction models showed that genotype moderately improved prediction of 5-HTT in caudate, but effects were not statistically significant after adjustment for multiple comparisons. Our observations provide additional evidence that serotonin-related genetic variants modulate adult human brain serotonin neurotransmission., (© 2023. Springer Nature Limited.)

The serotonin system plays a critical role in the modulation of impulsive aggression. Although serotonin transporters (SERT) are key in modulating synaptic serotonin levels, few studies have investigated the role of SERT levels in human impulsive aggression. The aim of this study was to investigate whether brain SERT levels are associated with trait impulsive aggression. We included 148 healthy individuals (mean age 29.3 ± 13.0, range 18-80 years, 91 females) who had undergone positron emission positron (PET) examinations with the SERT tracer [ 11 C]DASB and filled in self-report questionnaires of trait aggression, trait impulsivity and state aggression. We evaluated the association between cerebral SERT binding (BP ND ) and trait impulsive aggression in a latent variable model, with one latent variable (LV SERT ) modelled from SERT BP ND in frontostriatal and frontolimbic networks implicated in impulsive aggression, and another latent variable (LV IA ) modelled from various trait measures of impulsivity and aggression. The LV SERT was not significantly associated with the LV IA (p = 0.8). Post-hoc univariate analyses did not reveal any significant associations between regional SERT levels and trait aggression, trait impulsivity or state aggression, but we found that state aggression at the day of PET scan was significantly lower in L A /L A homozygotes vs S-carriers of the 5-HTTLPR gene (p = 0.008). We conclude that brain SERT binding was not related to variations in trait impulsive aggression or state aggression. Our findings do not support that SERT is involved in mediating the serotonergic effects on aggression and impulsivity, at least not in individuals with non-pathological levels of impulsive aggression., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest of relevance for the manuscript., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background . Chronic foot ulcers are a major cause of morbidity in people with diabetes with a lifetime risk of 25%. Treatment is challenging and the recurrence rates of foot ulcers are >50% after 3 years. Vitamin D deficiency is more common in people with diabetes with chronic foot ulcers, compared to both people without diabetes as well as people with diabetes but without foot ulcers. Purpose/aim of study . To assess the efficacy of high-dose compared to low-dose Cholecalciferol vitamin D3 on healing of chronic diabetic foot ulcers. Materials and methods . We included people with diabetes with one or more foot ulcers lasting for more than 6 weeks. Patients were randomly allocated to either a daily oral intake of high-dose (170 μg) or low-dose (20 μg) vitamin D3 (Cholecalciferol). We saw patients in the outpatient clinic after 4, 12, 24, 36, and 48 weeks. At each visit, we measured the ulcer with a specialized camera, and associated software and the area (cm 2 ) was calculated. Patients and assessors were blinded to treatment allocation. We followed all patients for 48 weeks or until wound healing or surgical treatment. Findings/results . We included 48 patients in the analysis (24 in each group), with a total of 64 ulcers. Among them 41 ulcers were followed until healing or 48-week follow-up and 20 ulcers were surgically treated during the study period. Three patients were lost for follow-up. The intention-to-treat analysis showed a significantly higher rate of ulcer healing in the high-dose group with 21 of 30 (70%) healed ulcers compared to 12 of 34 (35%) in the low-dose group ( P  = .012). Median ulcer reduction at final follow-up was 100% (interquartile range [IQR]: 72-100) in the high-dose group and 57% (IQR: -28 to 100) in the low-dose group. Furthermore, we found a significant effect of high-dose vitamin D on ulcer reduction in the repeated measures analysis of variance. Conclusions . We found high-dose vitamin D3 to be efficient, compared to low-dose vitamin D3, in promoting healing in chronic diabetic foot ulcers.

Ceftolozane-tazobactam is a new β-lactam/β-lactamase inhibitor combination approved by the U.S. Food and Drug Administration in 2019 for the treatment of hospital-acquired and ventilator-associated pneumonia. The combination is a particularly potent inhibitor of penicillin-binding proteins with higher affinity than other β-lactam agents. Persons with cystic fibrosis (pwCF) often harbour resistant Gram-negative bacteria in the airways and need antibiotics to prevent declining lung function. To test whether the introduction of ceftolozane-tazobactam in the period 2015-2020 led to a bacterial population level increase in cephalosporin resistance in a Danish CF population. In vitro, activity of ceftolozane-tazobactam was evaluated by susceptibility testing of clinical Pseudomonas aeruginosa isolated from pwCF from January 1, 2015, to June 1, 2020. Six thousand three hundred thirty two isolates collected from 210 adult pwCF were included. Thirty pwCF were treated with ceftolozane-tazobactam at least once. Ceftolozane-tazobactam exposure did not increase cephalosporin resistance on an individual or population level. However, resistance to ceftolozane-tazobactam was recorded despite no prior exposure in four pwCF. Compared to ceftazidime, ceftolozane-tazobactam had a better in vitro activity on P. aeruginosa. The percentage of non-mucoid P. aeruginosa isolates susceptible to ceftolozane-tazobactam were higher or equal to 5 other β-lactams. Ceftolozane-tazobactam expands the armamentaria against P. aeruginosa with acceptable levels for a selection of drug resistance., (© 2023 The Authors. APMIS published by John Wiley & Sons Ltd on behalf of Scandinavian Societies for Pathology, Medical Microbiology and Immunology.)

Purpose: Complication rates after fast-track optimization in bariatric surgery are varying. The aim of this study was to identify short-term complications in patients undergoing laparoscopic sleeve gastrectomy (SG) in an ERABS (enhanced recovery after bariatric surgery) optimized setup., Materials and Methods: This study is an observational analysis of a consecutive cohort of 1600 patients undergoing SG at an ERABS-optimized, private hospital during 2020 and 2021. Primary outcomes were length of stay, mortality, readmissions, reoperations, and complications according to the Clavien-Dindo classification (CDC) within postoperative day (POD) 30 and 90. Secondary outcomes were weight loss and quality of life (QoL) according to Moorehead-Ardelt questionnaires during the first postoperative year., Results: Primary outcomes: 99.1% of patients were discharged within POD 1. The 90-day mortality rate was zero. There were 1% readmissions and 1.2% reoperations within POD 30. Total 30-day complication rate was 4.6%, where 3.4% accounted for CDC grades ≤ II, and 1.3% accounted for CDC grade III. There were zero grade IV-V complications., Secondary Outcomes: One year after surgery, weight loss was substantial (p < 0.001), with an excess weight loss of 71.9%, and QoL had significantly increased (p < 0.001)., Conclusion: This study demonstrates that the use of an ERABS protocol in bariatric surgery does not compromise neither safety nor efficacy. Complication rates were low, and weight loss was significant. This study thus provides strong arguments that ERABS programs are beneficial in bariatric surgery., (© 2023. The Author(s).)

The microbiological quality of water is usually assessed by fecal coliform bacteria, and the presence of E. coli as an indicator of fecal contamination is widely recommended by international guidelines. This study aimed to assess the prevalence of diarrheagenic pathogens, in both public and personal domain water sources and examine the reliance on the WHO drinking water risk assessment guidelines. This study was conducted in a low-income urban community in Dhaka, Bangladesh between September 2014 and October 2015. Polymerase chain reaction (PCR) was used to detect the marker and virulence genes of Escherichia coli , Vibrio cholerae , Salmonella species, and Campylobacter species, and the culture method was employed for the quantitative assessment of E. coli . According to the WHO guidelines, 48% of the public domain source water and 21% of the personal domain point-of-drinking water were classified in the low-risk group, i.e., 0 CFU of E. coli /100 mL. However, when using PCR, we detected pathogens in 39% (14/36) of the point-of-drinking water samples and 65% (74/114) of the public domain water source samples classified in the low-risk group. Our study showed that relying solely on E. coli detection as a measure of water quality may overlook the presence of other pathogens in the drinking water. In addition to the culture-based method, the detection of virulence genes by PCR should also be considered to add more scrutiny to the detection of diverse types of pathogens.

Background: Up to 40% of adults over 65 years are full-time users of absorbent incontinence pads due to urinary incontinence. Simultaneously, urinary tract infection is amongst the most common hospital-acquired infection in older patients., Objectives: To explore the association between (1) full-time use of absorbent incontinence pads and urinary tract infection at acute hospital admission, (2) state of frailty and becoming a pad user during hospitalization, and (3) becoming a pad user and acquiring a urinary tract infection during hospitalization in older patients., Design: A retrospective cohort study., Setting: Admissions in an emergency department with transfers to geriatric, cardiac, infectious, or endocrinological wards from September 7th, 2017 to February 18th, 2019., Patients: 1,958 patients aged 65 years or more, having daily homecare or moderate comorbidity, hospitalized due to acute illness, and living in the municipality of Aarhus., Methods: The study was conducted by two researchers reviewing the patients' electronic health records combined with data on frailty status from a geriatric quality database. In the electronic health records, data on baseline characteristics, absorbent incontinence pad use at admission and during the hospital stay, and urinary tract infection were obtained., Results: Full-time users of absorbent incontinence pads had a higher probability of being admitted with urinary tract infection (Odds Ratio=2.00 (95% Confidence Interval: 1.61-2.49); p <.001). Patients identified as severely frail had a higher probability of becoming pad users during hospitalization (Odds Ratio=1.57 (95% Confidence Interval: 1.45-1.71); p <.001) compared to non/mild/moderate frail patients. Patients who became pad users during hospitalization had a higher risk of a hospital-acquired urinary tract infection (Odds Ratio=4.28 (95% Confidence Interval: 1.92-9.52); p <.001)., Conclusions: There was an association between the use of absorbent incontinence pads and the development of urinary tract infections in older hospitalized patients, both in full-time users and those who were frail and became pad users during hospitalization. These findings emphasize the need for further research on preventing urinary tract infections and unnecessary pad use in older patients., Competing Interests: None, (© 2023 The Authors. Published by Elsevier Ltd.)

Aim: There is need of an objective "standard procedure" that is reliable and clinically applicable for estimating oral neutrophil content in relation to oral diseases., Methods: Forty-one patients with suspected oral candidosis (OC) and nine healthy controls with no oral mucosal disease were flushing with 10 ml mouth rinse (MR) (sterile phosphate-buffered saline) for 1 min. Aliquots were stored on different conditions to explore stability, storage, and fixation conditions for analysis by flow cytometry., Results: The optimal storage and fixation condition for MR was by fixation 1 : 1 in 10% formalin and stored at 5°C. This procedure yielded stable results up to 7 days after collection. The ability of the optimized method to relate oral neutrophils to inflammation was demonstrated by the significantly higher number of neutrophils in patients with primary OC ( p = 0.0334) compared to healthy controls., Conclusion: This method is rapid, reliable, and clinically applicable for establishing the content of oral neutrophils. We demonstrate increased density of oral neutrophils in the MR of patients with OC. The potential of the method is to be "the standard procedure" for investigation of the oral inflammation in patients with oral diseases as it is noninvasive and provides high stability, clinical relevance, and minimal handling., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2023 Peter Østrup Jensen et al.)

The water sanitation and hygiene (WASH) sector has provided beneficiaries in low and middle-income countries with latrines and clean water for decades. However, we still need good evidence documenting the expected health impact. This paper investigates why we lack this evidence and ways to move forward. Using mTEC agar, we monitored E. coli contamination on selected "hotspot" surfaces within the kitchen environments of 32 low-income households in Dhaka, Bangladesh, every six weeks for two years. Despite being washed, the highest average contamination was found on food plates, at 253 cfu/10 cm 2 , followed by cutting knives, with 240 cfu/10 cm 2 . The drinking vessel surfaces and the latrine doorknobs had the lowest contaminations, with E. coli means of 167 and 73 cfu/10 cm 2 , respectively. These findings imply a need to measure an individual's pathogen exposure as close to the mouth as possible to estimate the true pathogen exposure. The paper proposes introducing the new "personal domain"-the point of consumption-as the physical sphere in which WASH interventions should be assessed. With this approach, we can observe and quantify the different pathogen exposure routes and, with this, further improve WASH interventions.

A growing interest in alternative cements has emerged with the sole purpose of reducing the environmental footprint associated with cement production. One of the promising alternatives is to use non-carbonate materials such as alkali-activated materials. They have demonstrated to have a similar performance as traditional Portland cement and have the potential to significantly reduce CO 2 emissions. This paper reviews the main relevant technologies that are already available in the construction industry and explains how to consider them for alkali-activated cement and concrete production. This includes aluminosilicate pre-treatment methods (drying, grinding, and calcining) to increase the precursor's reactivity and degree of amorphization, alkali activation by two-part or one-part mix, as well as, mixing and casting fresh alkali-activated concrete ensuring low porosity and adequate strength development. This review also presents an overview of the alkali-activated cements market, providing examples of commercialized products, estimating related CO 2 and costs, as well as future considerations for standardization and commercialization. Most of the commercialized alkali-activated materials are two-part mixes despite their limitations for in-situ applications. CO 2 emissions can be reduced by more than 68% when compared to Portland cements. However, they have been estimated to be 2 to 3 times more expensive and the cost is primarily dependent on the aluminosilicate and alkali activators source., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors.)

Objectives: To examine valsartan, losartan and irbesartan usage and switching patterns in the USA, UK, Canada and Denmark before and after July 2018, when the first Angiotensin-Receptor-Blocker (ARB) (valsartan) was recalled., Design: Retrospective cohort study., Setting: USA, Canadian administrative healthcare data, Danish National Prescription Registry and UK primary care electronic health records., Participants: Patients aged 18 years and older between January 2014 and December 2020., Intervention: Valsartan, losartan and irbesartan., Main Outcome: Monthly percentages of individual ARB episodes, new users and switches to another ARB, ACE inhibitors (ACEI) or calcium channel blockers containing products., Results: We identified 10.8, 3.2, 1.8 and 1.2 million ARB users in the USA, UK, Canada and Denmark, respectively. Overall proportions of valsartan, losartan and irbesartan use were 18.4%, 67.9% and 5.2% in the USA; 3.1%, 48.3% and 10.2% in the UK, 16.3%, 11.4% and 18.3% in Canada, 1%, 93.5% and 0.6% in Denmark. In July 2018, we observed an immediate steep decline in the proportion of valsartan use in the USA and Canada. A similar trend was observed in Denmark; however, the decline was only minimal. We observed no change in trends of ARB use in the UK. Accompanying the valsartan decline was an increase in switching to other ARBs in the USA, Canada and Denmark. There was a small increase in switching to ACEI relative to the valsartan-to-other-ARBs switch. We also observed increased switching from other affected ARBs, losartan and irbesartan, to other ARBs throughout 2019, in the USA and Canada, although the usage trends in the USA remained unchanged., Conclusion: The first recall notice for valsartan resulted in substantial decline in usage due to increased switching to other ARBs. Subsequent notices for losartan and irbesartan were also associated with increased switching around the time of the recall, however, overall usage trends remained unchanged., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

We explore the role of elites for development and the spread of industrialized dairying in Denmark in the 1880s. We demonstrate that the location of early proto-modern dairies, introduced by landowning elites from northern Germany in the eighteenth century, explains the location of industrialized dairying in 1890: an increase of one standard deviation in elite influence increases industrialized dairying by 56 percent of the mean exposure in one specification. We interpret this as evidence for a spread of ideas from the elites to the peasantry, which we capture through measures of specialization in dairying and demand for education and identify a causal relationship using an instrument based on distance to the influential first mover. Finally, we demonstrate that areas with cooperatives enjoyed greater wealth by the twentieth century, and that they are today associated with other Danish cultural attributes: a belief in democracy and individualism., Supplementary Information: The online version contains supplementary material available at 10.1007/s10887-023-09226-8., (© The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2023, Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.)

In vitro , cell-based assays are essential in diagnostics and drug development. There are ongoing efforts to establish new technologies that enable real-time detection of cell-drug interaction during culture under flow conditions. Our compact (10 × 10 × 8.5 cm) cell culture and microscope on disc (CMoD) platform aims to decrease the application barriers of existing lab-on-a-chip (LoC) approaches. For the first time in a centrifugal device, (i) cells were cultured for up to six days while a spindle motor facilitated culture medium perfusion, and (ii) an onboard microscope enabled live bright-field imaging of cells while the data wirelessly transmitted to a computer. The quantification of cells from the acquired images was done using artificial intelligence (AI) software. After optimization, the obtained cell viability data from the AI-based image analysis proved to correlate well with data collected from commonly used image analysis software. The CMoD was also suitable for conducting a proof-of-concept toxicity assay with HeLa cells under continuous flow. The half-maximal inhibitory time (IT50) for various concentrations of doxorubicin (DOX) in the case of HeLa cells in flow, was shown to be lower than the IT50 obtained from a static cytotoxicity assay, indicating a faster onset of cell death in flow. The CMoD proved to be easy to handle, enabled cell culture and monitoring without assistance, and is a promising tool for examining the dynamic processes of cells in real-time assays.

White alfalfa protein concentrate from alfalfa ( Medicago sativa ) is a promising substitute for milk and egg protein due to its functionality. However, it contains many unwanted flavours that limits the amount that can be added to a food without affecting its taste negatively. In this paper, we have demonstrated a simple method for the extraction of white alfalfa protein concentrate followed by a treatment with supercritical CO 2 . Two concentrates were produced at lab scale and pilot scale, with yields of 0.012 g (lab scale) and 0.08 g (pilot scale), of protein per g of total protein introduced into the process. The solubility of the protein produced at lab scale and pilot scale was approximately 30% and 15%, respectively. By treating the protein concentrate at 220 bar and 45 °C for 75 min with supercritical CO 2 , off-flavours were lowered. The treatment did not decrease the digestibility or alter the functionality of white alfalfa protein concentrate when it was used to substitute egg in chocolate muffins and egg white in meringues.

Importance: The direct and indirect implications of the COVID-19 pandemic have been associated with the mental health of children and adolescents, but it is uncertain whether these implications have been associated with changes in prescribing and diagnosis patterns., Objective: To examine psychotropic medication use and rates of psychiatric disorders in Danish children, adolescents, and young adults during the COVID-19 pandemic., Design, Setting, and Participants: This population-based, descriptive register-based cohort study included all Danish individuals aged 5 to 24 years from January 1, 2017, until June 30, 2022., Main Outcomes and Measures: Rates of filled prescriptions of psychotropic medications, including antipsychotics, anxiolytics, hypnotics, sedatives, antidepressants, and psychostimulants, and all inpatient and outpatient contacts with mental and behavioral disorders. Rates of new (incident) and total (prevalent) psychotropic medication use and psychiatric diagnoses were estimated. Rate ratios (RRs) were assessed between observed and expected numbers of incident psychotropic medication use or psychiatric diagnoses from March 2020 to June 30, 2022, comparing observed numbers with expected numbers predicted from the modeled prepandemic trend., Results: The study identified 108 840 (58 856 female individuals [54%]; median [IQR] age, 18 [14-22] years) incident psychotropic medication users. From March 2020 (first national lockdown) to June 2022, the rate of incident users of any psychotropic medication showed a relative increase of 18% (RR, 1.18; CI, 1.17-1.20) compared with expected numbers, which was primarily associated with an increase among those aged 12 to 17 years of 37% (RR, 1.37; 95% CI, 1.34-1.41). Similarly, there was an overall relative increase of incident psychiatric disorders of 5% (incidence rate, 1.05; CI, 1.04-1.07) (incident cases, 114 048 [58 708 female individuals (51%)]), which was associated with an increase in hyperkinetic disorders (RR, 1.13; CI, 1.09-1.18) and anxiety disorders (RR, 1.04; CI, 1.02-1.06). Prevalence patterns showed similar trends of an overall increase in psychotropic medication use and psychiatric disorders. One of 3 new users of an individual drug group had filled a prescription for a drug from another psychotropic medication group within the prior 6 months., Conclusions and Relevance: The results of this cohort study suggest that Danish youths experienced an increase in rates of psychotropic treatment and psychiatric disorder diagnoses during the COVID-19 pandemic, which was most pronounced among those aged 12 to 17 years. The increase was observed for children and adolescents with and without a psychiatric history within the last 5 years.

Introduction: Safe and effective pharmacological treatment is of paramount importance for treating severe psoriasis. Brodalumab, a monoclonal antibody against interleukin (IL) 17 receptor A, was granted marketing authorisation in the EU in 2017. The European Medicines Agency requested a postauthorisation safety study of brodalumab to address potential safety issues raised during drug development regarding major adverse cardiovascular events, suicidal conduct, cancer and serious infections., Methods and Analysis: BRodalumab Assessment of Hazards: A Multinational Safety is a multicentre observational safety study of brodalumab running from 2017 to 2029 using population-based healthcare databases from Denmark, Sweden, Norway, Netherlands, Germany and three different centres in Italy. A distributed database network approach is used, such that only aggregate data are exchanged between sites.Two types of designs are used: a case-time-control design to study acute effects of transient treatment and a variation of the new user active comparator design to study the effects of transient or chronic treatment. As comparators, inhibitors of TNF-α, inhibitors of IL-12 and IL-23, and other inhibitors of cytokine IL-17A are included.In the self-controlled case-time-control design, the risk of developing the outcome of interest during periods of brodalumab use is compared within individuals to the risk in periods without use.In the active comparator cohort design, new users of brodalumab are identified and matched to new users of active comparators. Potential baseline confounders are adjusted for by using propensity score modelling. For outcomes that potentially require large cumulative exposure, an adapted active comparator design has been developed., Ethics and Dissemination: The study is approved by relevant authorities in Denmark, Norway, Sweden, the Netherlands, Germany and Italy in line with the relevant legislation at each site. Data confidentiality is secured by the distributed network approach. Results will be published in peer-reviewed journals., Trial Registration Number: EUPAS30280., Competing Interests: Competing interests: MR and PBJ report participation in research funded by LEO Pharma A/S (no personal fees). participation in studies funded by Alcon, Almirall, Astellas, AstraZeneca, Boehringer-Ingelheim, Novo Nordisk, Servier, Pfizer, Menarini Pharmaceuticals, and LEO Pharma, all regulator mandated phase IV studies, all with funds paid to the institution (no personal fees). JH has received teaching fees from Atrium. HS reports participation in research funded by LEO Pharma A/S (no personal fees). He has received personal consulting fees from Bristol-Myers-Squibb, Novartis, Roche, Merck and Pfizer outside the submitted work. He has received teaching fees from Atrium. KF and ØK reports participation in research projects funded by Novo Nordisk and LEO Pharma, all regulator mandated phase IV studies, all with funds paid to the institution (no personal fees). ES, EH and RH are employees of the PHARMO Institute for Drug Outcomes Research. This independent research institute performs financially supported studies for government and related healthcare authorities and several pharmaceutical companies. AE has received research funding from Pfizer, Eli Lilly, Novartis, Bristol-Myers Squibb, AbbVie, Janssen Pharmaceuticals, the Danish National Psoriasis Foundation, the Simon Spies Foundation and the Kgl Hofbundtmager Aage Bang Foundation, and honoraria as consultant and/or speaker from AbbVie, Almirall, Leo Pharma, Zuellig Pharma, Galápagos NV, Sun Pharmaceuticals, Samsung Bioepis, Pfizer, Eli Lilly and Company, Novartis, Union Therapeutics, Galderma, Dermavant, UCB, Mylan, Bristol-Myers Squibb, Horizon Therapeutics and Janssen Pharmaceuticals. UK, Alessandro Cesare Rosa, and Valeria Belleudi are employees of the Department of Epidemiology ASL Roma1, Lazio Regional Health Service in Rome, Italy. Our publicly funded department performs epidemiological research and receives grants from public national and international organisations. In case of financial support from private organisations, an ethical code of conduct is signed to guarantee transparency and independence. KG, DH and JR are employees of the Centre for Pharmacoepidemiology of the Karolinska Institutet in Sweden, which receives grants from regulatory authorities, pharmaceutical companies, and contract research organisations for performance of drug safety and drug utilisation studies. UH, WS and SS are working at an independent, non-profit research institute, the Leibniz Institute for Prevention Research and Epidemiology-BIPS. Unrelated to this study, BIPS occasionally conducts studies financed by the pharmaceutical industry. Almost exclusively, these are postauthorisation safety studies (PASS) requested by health authorities. GT declares participation to advisory boards on topics not related to this paper and sponsored by the following pharmaceutical companies in the last two years: Eli Lilly; Sanofi; Amgen; Novo Nordisk; Sobi; Gilead; Celgene; Daikii Sankyo. RG is employed by ARS, a public research centre participating in pharmacoepidemiology studies funded by public or private institutions and compliant with the ENCePP Code of Conduct. The budget of ARS is partially sustained by such studies. Lise Skov Ranch is a full time employee of LEO Pharma A/S., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Most cholera outbreaks in Bangladesh are seasonal, peaking in the dry and post-monsoon periods. Therefore, we investigated whether changes in water, sanitation, and hygiene (WASH) behavior in three populations in Bangladesh during the year could help explain why these two periods are particular to cholera transmission. The study used a mixed-method design, including a repeated cross-sectional study, focus group discussions, and key informant interviews. Through a repeated cross-sectional study, WASH-related variables were assessed during the dry, monsoon, and control seasons in 600 households from coastal Satkhira, inland Sirajganj, and the Dhaka slums. Seasonal behavioral changes were observed in all study areas. Dhaka and Satkhira had an increased mean distance to water sources during the dry and monsoon seasons (Dhaka: control season, 12 m [95% CI, 11-13]; dry season, 36 m [95% CI, 18-55]; and monsoon season, 180 m [95% CI, 118-243]; Satkhira: control season, 334 m [95% CI, 258-411]; dry season, 669 m [95% CI, 515-822]; and monsoon season, 2,437 m [95% CI, 1,665-3,209]). The participants attributed this to pollution of the usual water source. Perceived water quantity was lowest during the dry season in Dhaka and Sirajganj, and during the monsoon season in Satkhira. Handwashing with soap declined in all areas during the dry and monsoon seasons. Open defecation was frequent among children younger than 5 years, increasing during seasonal climate hazards. Results show that WASH-related behavior changed seasonally, increasing the risk of cholera transmission through multiple hygiene-related transmission pathways. Future research would benefit by ensuring that the length of studies covers all seasons throughout the year and also by looking in more detail at people's behavior and hygiene practices.

The cortisol awakening response (CAR) describes the sharp increase in cortisol secretion within 60 min after awakening. A summary of the CAR, the area under the cortisol curve above the awakening cortisol value (AUC i ) is a widely used biomarker in health research. Estimation of the AUC i rely on a number of collected salivary samples at fixed time intervals (i.e., 5 samples in 15 min intervals) starting from awakening. Little empirical work has been executed to investigate the impact of reducing sampling times on AUC i estimation, which could potentially improve participant compliance and reduce operational costs. This study aimed to assess the reliability and validity of using 3-sample AUC i versus 5-sample AUC i , i.e., systematic and random fluctuations based on a large dataset from healthy and case individuals (total n = 537). We showed that the ideal timing of 3-sampling times was 0-30-60 min with a median difference in AUC i of - 8 nmol*h/L and interquartile range of 65 nmol*h/L among healthy individuals, and - 12 nmol*h/L and 78 nmol*h/L among case individuals. We subsequently validated the 3-sample AUC i by re-analyzing three published association studies. Overall, we obtained similar p-values with 3-sample AUC i when compared to 5-sample AUC i , while smaller effect sizes and standard errors were observed. In conclusion, despite a less precise estimation of the AUC i itself, our data support that the AUC measure of the CAR, based on three samples collected at 0-30-60 min from awakening, provides reliable results in association studies., Competing Interests: Conflict of interest AN, BO, ESH and PSJ declare no conflicts of interest. VGF declares that she has received honorarium for advisory meetings and lectures for Sage Therapeutics and H. Lundbeck., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Introduction: Chronic wounds have a compromised microcirculation which leads to restricted gas exchange. The majority of these hypoxic wounds is infested with microorganisms congregating in biofilms which further hinders the antibiotic function. We speculate whether this process can be counteracted by hyperbaric oxygen therapy (HBOT)., Methodology: Twenty-eight BALB/c mice with third-degree burns were included in the analyses. Pseudomonas aeruginosa embedded in seaweed alginate beads was injected under the eschar to mimic a biofilm infected wound. Challenged mice were randomized to receive either 4 days with 1 x ciprofloxacin combined with 2 × 90 min HBOT at 2.8 standard atmosphere daily, 1 x ciprofloxacin as monotherapy or saline as placebo. The mice were clinically scored, and wound sizes were estimated by planimetry daily. Euthanasia was performed on day 8. Wounds were surgically removed in toto , homogenized and plated for quantitative bacteriology. Homogenate supernatants were used for cytokine analysis., Results: P. aeruginosa was present in all wounds at euthanasia. A significant lower bacterial load was seen in the HBOT group compared to either the monotherapy ciprofloxacin group (p = 0.0008), or the placebo group (p < 0.0001). IL-1β level was significantly lower in the HBOT group compared to the placebo group (p = 0.0007). Both treatment groups had higher osteopontin levels than the placebo group (p = 0.002 and p = 0.004). The same pattern was seen in the S100A9 analysis (p = 0.01 and p = 0.008), whereas no differences were detected between the S100A8, the VEGF or the MMP8 levels in the three groups., Conclusion: These findings show that HBOT improves the bactericidal activity of ciprofloxacin against P. aeruginosa wound biofilm in vivo. HBOT in addition to ciprofloxacin also modulates the host response to a less inflammatory phenotype., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Claus Moser reports financial support was provided by 10.13039/501100009708Novo Nordisk Foundation., (© 2023 The Authors.)

Lyme neuroborreliosis (LNB), a tick-borne infection caused by spirochetes within the Borrelia burgdorferi sensu lato (s.L.) complex, is among the most prevalent bacterial central nervous system (CNS) infections in Europe and the US. Here we have screened a panel of low-passage B . burgdorferi s.l. isolates using a novel, human-derived 3D blood-brain barrier (BBB)-organoid model. We show that human-derived BBB-organoids support the entry of Borrelia spirochetes, leading to swelling of the organoids and a loss of their structural integrity. The use of the BBB-organoid model highlights the organotropism between B . burgdorferi s.l. genospecies and their ability to cross the BBB contributing to CNS infection., Competing Interests: Disclosures: Outside of the present work: A.M.L. reports speaker’s honorarium/travel grants and advisory board activity from Gilead, GSK, and Pfizer. The other authors declare no competing interests exist. P.E.L. has been an external scientific expert to Valneva Austria GmbH and Pfizer Inc. and received speaker’s honorarium and travel grants., (© 2022.)

There is a paucity of recent research on direct water quantity measurement for personal and domestic hygiene. We aimed to measure the water quantity used for personal and domestic hygiene and to explore the reasons and determinants for variation of water usage. We conducted this study from September 2014 to June 2016 in a low-income urban community in Dhaka. In 12 households, the team conducted a day-long bimonthly ethnographic observation for one year to measure the volume of water used per activity per person. They conducted 28 in-depth interviews to explore the reasons for changes of water usage. Participants used a median of 75 L (61-100) of water per capita per day (LCPD) and of this 75 LCPD they used a median of 39 LCPD (26-58) for personal hygiene. Women used less water than men. Individual and social norms, beliefs, and weather determinants determined personal hygiene. Water availability determined domestic hygiene (e.g., washing dishes, toilets and bathrooms). This study helps to elucidate a range of determinants of water usage of the participants from the participants' perspective. The quantity of water used for domestic and personal hygiene and its relationship to fecal-oral transmitted disease can be explored in future research.

Objectives: Reactivation of HERV-K(HML-2) has been found in subsets of individuals with amyotrophic lateral sclerosis (ALS). This study examines the antibody response against HML-2 in ALS and analyzes its clinical relevance., Methods: Antibodies to HML-2 envelope (env) were analyzed using a peptide array for epitope mapping and by a peptide enzyme-linked immunosorbent assay (ELISA) in 242 healthy donors, and 243 ALS and 85 multiple sclerosis (MS) individuals. Extracellular levels of HML-2 were analyzed by digital polymerase chain reaction (PCR)., Results: Antibodies in the sera of ALS individuals recognized more HML-2 env peptides compared to healthy controls (p < 0.0001). ALS individuals had higher levels of HML-2 than healthy donors (p = 0.02) and higher antibody levels against a select HML-2 env peptide compared to healthy donors or individuals with multiple sclerosis (p < 0.0001). 55.14% of ALS compared to 21.16% of healthy donors and 13.10% of MS individuals had antibodies against the HML-2 peptide (AUC = 0.769, p < 0.0001). Levels of extracellular HML-2 DNA in serum (p = 0.02) and the number of HML-2 env peptides recognized by ALS sera (p = 0.02) correlated with disease duration. Among ALS individuals, lower levels of HML-2 antibodies were associated with a definite diagnosis per EL Escorial criteria (p = 0.03), and with a lower predicted (p = 0.02) and observed survival (p = 0.03)., Interpretation: There is a differential antibody response against specific epitopes of HML-2 env in ALS and controls, suggesting epitope spreading, likely due to persistent antigenic exposure following reactivation of the viral genes. Low levels of antibodies to HML-2 env in ALS are associated with poor prognosis and decreased survival probability. ANN NEUROL 2022;92:782-792., (© 2022 The Authors. Annals of Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.)

The potentially deadly and sporadic diarrhea-causing agent, Vibrio cholerae , is present in a great number in the freshwater aquatic environment and can be transmitted to humans by different aquatic organisms. In the perspective of Bangladesh, an anadromous fish species Hilsha ( Tenualosa ilisha ) can act as a transmission vehicle of V. cholerae from the aquatic to the household kitchen environment. The present study was carried out to investigate the presence of V. cholerae in the aquatic habitat of Bangladesh with a major emphasis on freshly caught Hilsha fish, along with river water and plankton samples from the fish capture site. The study also detected the biofilm formation capability of V. cholerae within Hilsha fish that might help the transmission and persistence of the pathogen in aquatic habitat. Twenty out of 65 freshly caught fish (30.8%) and 1 out of 15 water samples (6.67%) showed the presence of V. cholerae and none of the plankton samples were positive for V. cholerae . The isolated strains were identified as non-O1 and non-O139 serogroups of V. cholerae and contain some major toxin and virulence genes. A few strains showed cellular cytotoxicity on the HeLa cell line. All strains were able to form biofilm on the microtiter plate and the detection of three genes related to biofilm formation ( vpsA , vpsL , and vpsR ) were also assayed using qPCR. In this study, the in vitro biofilm formation ability of the isolated strains may indicate the long-term persistence of V. cholerae in different parts of Hilsha fish. The abundance of V. cholerae only in freshly caught Hilsha fish and the absence of the pathogen in the surrounding aquatic environment could stipulate the role of Hilsha fish as one of the major transmission routes of V. cholerae from the freshwater aquatic environment of Bangladesh to the household kitchen environment., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Dey, Hossain, Akhter, Jensen and Begum.)

As a consequence of the increased demand for proteins for both feed and food, alternative protein sources from green plants such as alfalfa ( Medicago sativa ) have come into focus, together with methods to recover these proteins. In this study, we have investigated the use of screw presses for protein recovery from alfalfa at laboratory and pilot scale. We found that using a pilot scale screw press, with a working pressure of 6 bar, 16% of the total protein was recovered in one pressing, and that after rehydrating and repressing the alfalfa up to ten times, 48% of the total protein could be recovered. The green alfalfa protein concentrate was analyzed for total protein, amino acid profile, protein digestibility, color, ash, fiber and fat content. It was found that repetitive pressings lowered the digestibility of the protein pool and reduced the total protein concentration due to dilution. To achieve the best quality protein at the highest concentrations, it is recommended to press the alfalfa no more than twice, which results in an alfalfa protein concentrate with more than 32% soluble protein and greater than 82% digestibility.

This study aims to improve the understanding of the formation and prevention of deposits caused by the introduction of alternative fuels in the cement industry. Experiments were conducted with cement mineral materials in a laboratory-scale entrained flow reactor. To simulate the temperature conditions in a cement calciner, two different deposit probe systems were used: a high probe surface temperature (HPST) deposit system with a probe surface temperature range of 700-1200 °C and a low probe surface temperature (LPST) deposit system with a probe surface temperature range of 500-700 °C. The effects of fed materials (raw meal, hot meal, bypass dust), flue gas temperature (700-1200 °C), deposit probe surface temperature (550-1200 °C), gas velocity (0.9-2.7 m/s), and experimental duration (5-60 min) on the deposit formation rate were investigated. The results revealed that the concentration of KCl in the fed materials has a large influence on deposit formation. In HPST experiments with furnace temperatures ranging from 700 to 1100 °C, the bypass dust has a higher deposition rate than hot and raw meals, due to a large amount of K and Cl (>10 wt %) in the bypass dust, which forms melts and increases the stickiness between impacting particles and deposits. In LPST experiments, the presence of KCl facilitated the deposit formation rate by providing a sticky layer on the deposit probe via condensation, resulting in a higher deposition rate of bypass dust than the raw meal. An increase in gas velocity showed a negative effect on the deposit formation rate due to an increase in rebound. The present study suggests that keeping the calciner wall temperature at 900-1100 °C and a low level of KCl content would effectively reduce the deposit formation, thereby increasing the equipment life and reducing the operating cost during cement production., Competing Interests: The authors declare no competing financial interest., (© 2022 The Authors. Published by American Chemical Society.)

Objective: Magnetic resonance imaging (MRI)-based techniques for non-invasive assessing liver iron concentration (LIC) in patients with iron overload have a limited upper measuring range around 35 mg/g dry weight, caused by signal loss from accelerated T1-, T2-, T2* shortening with increasing LIC. Expansion of this range is necessary to allow evaluation of patients with very high LIC., Aim: To assess measuring range of a gradient-echo R2* method and a T1-weighted spin-echo (SE), signal intensity ratio (SIR)-based method (TE = 25 ms, TR = 560 ms), and to extend the upper measuring range of the SIR method by optimizing echo time (TE) and repetition time (TR) in iron-loaded minipigs., Methods: Thirteen mini pigs were followed up during dextran-iron loading with repeated percutaneous liver biopsies for chemical LIC measurement and MRIs for parallel non-invasive estimation of LIC (81 examinations) using different TEs and TRs., Results: SIR and R2* method had similar upper measuring range around 34 mg/g and similar method agreement. Using TE = 12 ms and TR = 1200 ms extended the upper measuring range to 115 mg/g and yielded good method of agreement., Discussion: The wider measuring range is likely caused by lesser sensitivity of the SE sequence to iron, due to shorter TE, leading to later signal loss at high LIC, allowing evaluation of most severe hepatic iron overload. Validation in iron-loaded patients is necessary., (© 2022. The Author(s).)

Background: A basic paradigm of human infection is that acute bacterial disease is caused by fast growing planktonic bacteria while chronic infections are caused by slow-growing, aggregated bacteria, a phenomenon known as a biofilm. For lung infections, this paradigm has been thought to be supported by observations of how bacteria proliferate in well-established growth media in the laboratory-the gold standard of microbiology., Objective: To investigate the bacterial architecture in sputum from patients with acute and chronic lung infections., Methods: Advanced imaging technology was used for quantification and direct comparison of infection types on fresh sputum samples, thereby directly testing the acute versus chronic paradigm., Results: In this study, we compared the bacterial lifestyle (planktonic or biofilm), growth rate and inflammatory response of bacteria in freshly collected sputum (n=43) from patient groups presenting with acute or chronic lung infections. We found that both acute and chronic lung infections are dominated by biofilms (aggregates of bacteria within an extracellular matrix), although planktonic cells were observed in both sample types. Bacteria grew faster in sputum from acute infections, but these fast-growing bacteria were enriched in biofilms similar to the architecture thought to be reserved for chronic infections. Cellular inflammation in the lungs was also similar across patient groups, but systemic inflammatory markers were only elevated in acute infections., Conclusions: Our findings indicate that the current paradigm of equating planktonic with acute and biofilm with chronic infection needs to be revisited as the difference lies primarily in metabolic rates, not bacterial architecture., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY. Published by BMJ.)

Chronic infections caused by microbial biofilms represent an important clinical challenge. The recalcitrance of microbial biofilms to antimicrobials and to the immune system is a major cause of persistence and clinical recurrence of these infections. In this Review, we present the extent of the clinical problem, and the mechanisms underlying the tolerance of biofilms to antibiotics and to host responses. We also explore the role of biofilms in the development of antimicrobial resistance mechanisms., (© 2022. Springer Nature Limited.)

This is a case report of a young woman with diabetes mellitus type 1. She was admitted with severe diabetic ketoacidosis. Asymptomatic "surgical emphysema", pneumomediastinum and bilateral pneumothoraces were accidently discovered. The emphysema had probably occurred due to laboured breathing and groaning during her diabetic ketoacidosis. No treatment was needed.

Pseudomonas aeruginosa is a human pathogen associated with both acute and chronic infections. While intensively studied, the basic mechanisms enabling the long-term survival of P. aeruginosa in the host, despite massive immune system attack and heavy antimicrobial treatment, remain to be identified. We argue that such infections may represent niche invasions by P. aeruginosa that influence the microenvironment by depleting host-derived substrate and activating the immune response. Bacteria embedded in cell aggregates establish a microenvironmental niche, where they endure the initial host response by slowing down their metabolism. This provides stable, lasting growth conditions with a constant, albeit slow supply of substrate and electron acceptors. Under such stable conditions, P. aeruginosa exhibits distinct adaptive traits, where its gene expression pattern reflects a life exposed to continuous attack by the host immune system and antimicrobials. Here, we review fundamental microenvironmental aspects of chronic P. aeruginosa infections and examine how their structural organization influences their in vivo microenvironment, which in turn affects the interaction of P. aeruginosa biofilm aggregates with the host immune system. We discuss how improving our knowledge about the microenvironmental ecology of P. aeruginosa in chronic infections can be used to combat persistent, hard-to-treat bacterial infections., (© The Author(s) 2022. Published by Oxford University Press on behalf of FEMS.)