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Charlotte Thor Petersen,1,2 Kristoffer Jarlov Jensen,1 Mary Rosenzweig,2 Benedikte Irene von Osmanski,1,2 Mikkel Zöllner Ankarfeldt,1 Janne Petersen1,3 1Copenhagen Phase IV Unit (Phase4CPH), Copenhagen University Hospital Bispebjerg and Frederiksberg, Copenhagen, Denmark; 2Life Science Insights Centre, DLI Market Intelligence, Copenhagen, Denmark; 3Section of Biostatistics, University of Copenhagen, Copenhagen, DenmarkCorrespondence: Charlotte Thor Petersen, Life Science Insights Centre, DLI Market Intelligence, Copenhagen, Denmark, Tel +45 30 35 20 43, Email ctp@dlimi.comPurpose: There is an increasing need for national and international pharmacoepidemiological studies based on high-quality real-world data of which the Danish registries are a valuable source. In lack of a complete overview of which data are used to assess real-world drug safety and effectiveness outcomes, we aimed to map the outcomes, data sources, and the reporting of outcome quality in recent pharmacoepidemiological studies.Methods: We conducted a systematic mapping review of pharmacoepidemiological studies based on Danish registries investigating drug safety and/or effectiveness, published in the period 2018– 2019, identified in PubMed and Scopus. Extraction included: Anatomical Therapeutic Chemical level 2 code for drug exposures, outcomes, outcome data sources, and quality of outcomes.Results: Of the 210 included studies, 96% used outcomes categorized as Clinical, 4% utilized outcomes categorized as Society-related, 5% used outcomes categorized as Healthcare cost and utilization, and 3% of the studies applied outcomes categorized as Patient-reported in which the percentages are not mutually exclusive. Diagnosis (66%) and Mortality (38%) were the two most utilized subcategories among those categorized as Clinical outcomes. Danish Health Data Authority and Statistics Denmark registries were the most reported outcome data sources (90%). Ninety-six studies (46%) reported one or more quality parameters related to their outcomes of interest with accuracy/validity being the most reported parameter (22%).Conclusion: The Danish registries support a wide range of outcomes. Across therapeutic areas, most studies investigate traditional clinical outcomes of disease and mortality based on data from a small number of available registries. In contrast, clinical and biochemical databases, despite potentially offering outcomes with high responsiveness, and the high-quality social and healthcare cost registries were rarely used as outcome data sources.Keywords: pharmacoepidemiology, registries, review, Denmark, drugs

Herein, we describe the design and synthesis of 16 neo-glycolipids that are potential permeation enhancers for oral drug delivery of peptide therapeutics. These amphiphilic neo-glycolipids are composed of fatty acids and various carbohydrates (d-glucose, lactose, cellobiose, maltose) via an oxime linker. The ability of the synthesized neo-glycolipids to enhance permeation of fluorescein-labelled dextran (4 kDa) or 3 H-mannitol across intestinal epithelium was investigated in vitro using monolayers of human epithelial Caco-2 cells. Their effects were compared with (pre-)clinically known enhancers as reference compounds; sodium salts of octanoic, decanoic, and dodecanoic acid, and sodium salcaprozate (SNAC). Most neo-glycolipids increased the permeation of the model compounds, proving that neo-glycolipids, which possess vastly different properties from the reference compounds, e. g., in terms of clogD and polar surface area, are effective permeation enhancers. The neo-glycolipid based on decanoic acid and glucose was more potent than related compounds based on disaccharides. Significant differences in solubility and cellular compatibility were found for neo-glyolipids based on different carbohydrates. Finally, neo-glycolipids were evaluated as permeation enhancers for the peptide hormone PYY 3-36 . Glucose- and maltose-derived neo-glycolipids based on decanoic and dodecanoic acid showed promising enhancements in PYY 3-36 permeation in vitro while maintaining good cellular compatibility, relevant for oral delivery of obesity treatments., (© 2024 Wiley-VCH GmbH.)

Peptides are highly efficient for treatment of many diseases, especially in oncology and diabetes. Oral delivery of peptides is desirable, but is challenged by low bioavailability and new chemical methods to enable oral delivery are needed. Here, we developed pH responsive linearization as a strategy for transient protection of peptides to extend their half-life in model systems. Peptides were cyclized to increase their stability at the low pH in the stomach, while they linearize at neutral pH to form the active peptide. We developed ester based responsive linkers with a protonable amine for O-to-N acyl shift, which allowed linearization strategies based on pyroglutamoyl (pGlu) or diketopiperazine (DKP) formation. After coupling of the linker, peptides were cyclized by CuAAC. We studied the stability against simulated gastric fluid (SGF) at different pH and the ability of cyclic peptides to linearize. This led to PYY3-36 analogues with pH responsive linearization for increased stability., (© 2024 Wiley‐VCH GmbH.)

Background: The beneficial effects of Bacillus Calmette-Guérin (BCG) as an intervention against non-mycobacterial infections have been extensively studied in randomized trials. These non-specific effects have been linked to a heterologous increase of pro-inflammatory cytokine production by innate immune cells. It is unknown if BCG induces such responses in older individuals from TB-endemic countries., Methods: In a single-blinded trial in Guinea-Bissau, 40 adults over 50 years of age were randomized 1:1 in a block of 40 to intradermal injection of BCG-Japan (intervention) or solvent (placebo). Production of interleukin (IL)-1β, IL-6, IL-10, interferon (IFN)-γ and tumor necrosis factor (TNF)-α was measured by ELISA in supernatant of peripheral blood mononuclear cells stimulated with Mycobacterium tuberculosis and heterologous pathogens. The trial was registered at clinicaltrials.gov (NCT02953327)., Findings: Between January 25 and March 7, 2017, 40 individuals were randomized. Two months after vaccination, BCG-Japan recipients (n = 11) had higher production of IFN-γ to M. tuberculosis stimulation (Geometric mean ratio (GMR): 3·91 [95 % Confidence Interval (CI), 1·53-9·96]) and increased release of the pro-inflammatory innate cytokines IL-1β, IL-6 and TNF-α to non-specific stimuli (GMR TNF-α: 1·47 [95 % CI, 0·98-2·19]) than their controls (n = 13). Both the specific and non-specific responses were more pronounced among those with a positive QuantiFERON at baseline., Interpretation: BCG-Japan can induce a trained immunity phenotype in older adults. These effects were particularly strong in previously M. tuberculosis exposed individuals. Future randomized trials are needed to determine BCG's potential to protect the older populations from infections-driven morbidity and mortality., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Christine Stabell Benn reports financial support was provided by Danish National Research Foundation. Christine Stabell Benn reports financial support was provided by European Research Council. Mihai Netea reports financial support was provided by European Research Council. Mihai Netea reports financial support was provided by Royal Netherlands Academy of Arts and Sciences. Christian Wejse reports a relationship with Danish National Board of Patient Safety that includes: consulting or advisory. Christian Wejse reports a relationship with Novartis that includes: speaking and lecture fees., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Chemical modification of proteins is of growing importance to generate new molecular probes for chemical biology and for the development of new biopharmaceuticals. For example, two approved, long-acting insulin variants are lipidated at the LysB29 side-chain. Acylations of proteins have so far been performed in batch-mode. Here we describe the use of flow chemistry for site-selective acylation of a small protein, insulin. To the best of our knowledge this is the first report on flow chemistry for chemical modification of insulin. The first step was to develop reaction conditions for acylation of Lys B29 that gave a soluble mixture and thus was compatible with flow chemistry in a microreactor; this included selection of a soluble base. Secondly, the conditions, such as reagent ratios and flow rate were optimized. Third, the use of these conditions for the acylation with a wide range of acids was demonstrated. Finally, Boc-protected insulins were synthesized. Insulin remained stable towards these flow chemistry conditions. This use of flow chemistry for the chemical modification of insulin opens the prospect of producing chemically modified biopharmaceuticals by flow chemistry with fewer byproducts., (© 2024 Wiley-VCH GmbH.)

Cerebellar abscess is a reported complication of chronic suppurative otitis media (CSOM) and is life threatening at times. It usually develops by direct spread of CSOM through the bony erosion in the petrous bone or by thrombophlebitis of the sigmoid sinus. However, an alternative pathway of infection transmission from the petrous bone to the cerebellum through the anatomical bridge of the cranial nerves has possibly not been described before. A 63-year-old female patient with CSOM and cholesteatoma developed ataxia and right facial palsy. Computed tomography (CT) showed bone erosion of the right petrous bone suggesting middle ear infection. Post-contrast MRI revealed an enhancement of swollen 7 th /8 th nerve complex, suggesting neuritis, and cholesteatoma in the right petrous bone. It also showed ring enhancing lesion in the cerebellar peduncle of the same side suggesting brain abscess. Surgical intervention was performed emergently and pus aspirated. She also underwent mastoidectomy and removal of cholesteatoma later by the otolaryngology team and finally got better. This case illustrates that CSOM can cause cerebellar abscess by spreading infection via anatomical bridge of the cranial nerves without direct invasion from the temporal bone or thrombophlebitis of sinus., Competing Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (© The Author(s) 2024.)

Background: Past and ongoing advancements in cystic fibrosis (CF) care warrant long-term analysis of the societal impact of the condition. This study aims to evaluate changes in key socioeconomic factors across three decades among people living with CF (pwCF), compared with both the general population and an early-onset chronic disease population., Methods: This nationwide, registry-based, matched cohort study included all pwCF ≥ 18 years in Denmark in the years 1990, 2000, 2010, and 2018. Each person living with CF was matched to five individuals in the general population and five individuals living with type 1 diabetes or juvenile arthritis based on age, sex, and municipality., Results: The Danish adult CF population increased nearly fourfold from 88 in 1990 to 331 in 2018, and mean age increased by ten years. The educational level of pwCF was similar to the two comparator cohorts, while pwCF were less often in employment and more often permanently outside the labor force. Personal and household income levels of the CF cohort were higher than those of the comparator cohorts., Conclusions: The disadvantage in employment for pwCF remained, but, over time, the societal profiles of the one-year CF cohorts increasingly converged with those of the comparator cohorts, indicative of improved clinical management, extended life expectancy, and the supportive role of the Danish welfare system in reducing health inequalities. Further research should be done to evaluate the effects of the newly introduced modulator therapies on employment, considering the broader societal impact and impact on quality of life., Competing Interests: Declaration of competing interest The research project is funded by Vertex Pharmaceuticals. All funds were given to the institutions of the investigators. The research was performed in collaboration with the sponsor., (Copyright © 2024. Published by Elsevier B.V.)

Persistent pain affects one in five people worldwide, often with severely debilitating consequences. Current treatment options, which can be effective for mild or acute pain, are ill-suited for moderate-to-severe persistent pain, resulting in an urgent need for new therapeutics. In recent years, the somatostatin receptor 4 (SSTR 4 ), which is expressed in sensory neurons of the peripheral nervous system, has emerged as a promising target for pain relief. However, the presence of several closely related receptors with similar ligand-binding surfaces complicates the design of receptor-specific agonists. In this study, we report the discovery of a potent and selective SSTR 4 peptide, consomatin Fj1, derived from extensive venom gene datasets from marine cone snails. Consomatin Fj1 is a mimetic of the endogenous hormone somatostatin and contains a minimized binding motif that provides stability and drives peptide selectivity. Peripheral administration of synthetic consomatin Fj1 provided analgesia in mouse models of postoperative and neuropathic pain. Using structure-activity studies, we designed and functionally evaluated several Fj1 analogs, resulting in compounds with improved potency and selectivity. Our findings present a novel avenue for addressing persistent pain through the design of venom-inspired SSTR 4 -selective pain therapeutics., One Sentence Summary: Venom peptides from predatory marine mollusks provide new leads for treating peripheral pain conditions through a non-opioid target.

Background: One-third of American adults encompassed by current colorectal cancer screening guidelines fail to obtain recommended screening evaluations. Educational videos are a valuable medium through which to educate and encourage recommended health behaviors in patients., Methods: A cross-sectional study reviewing the quality of patient education videos addressing colorectal cancer screening. Video quality was assessed in 3 domains: accountability, content, and production., Results: Forty-four videos met inclusion criteria. Out of 33 possible points, videos scored a median of 15.0 (interquartile range 12.9-16.6). Videos scored 1.0 (interquartile range .8-1.0) out of 4.0 for accountability, 6.0 (interquartile range 4.4-8.0) out of 20 for content, and 8.0 (interquartile range 7.4-8.0) out of 9.0 for production. Colonoscopy was the most frequently discussed method of screening (38, 86%). While 13 (34%) videos discussed the risk of colorectal cancer in the general population and 15 (32%) discussed the risk in those with a family history, few videos addressed those with other risk factors. Most (31, 70%) videos discussed the medical consequences of not receiving screening, but only 1 (2%) video discussed the social consequences. Similarly, medical benefits were discussed in 34 (77%) videos while other benefits were not discussed by any video. Only one-fifth of the videos address three or more barriers to screening., Conclusions: Videos on colorectal cancer screening have excellent production quality but need improvement in the domains of accountability and content. The videos included in this analysis did not adequately address the concerns of viewers nor the benefits of colorectal cancer screening., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Objective: This study aims to evaluate the developments in the testing of Kirsten Rat Sarcoma viral oncogene homolog (KRAS) and v-Raf murine sarcoma viral oncogene homolog B1 (BRAF) mutations across different cancer types and regions in Denmark from 2010 to 2022., Study Design and Setting: Using comprehensive data from the Danish health registries, we linked molecular test results from the Danish Pathology Registry with cancer diagnoses from the Danish National Patient Registry between 2010 and 2022. We assessed the frequency and distribution of KRAS and BRAF mutations across all cancer types, years of testing, and the five Danish regions., Results: The study included records of KRAS testing for 30 671 patients and BRAF testing for 30 860 patients. Most KRAS testing was performed in colorectal (78%) and lung cancer (18%), and BRAF testing in malignant melanoma (13%), colorectal cancer (67%), and lung cancer (12%). Testing rates and documentation mutational subtypes increased over time. Reporting of wildtype results varied between lung and colorectal cancer, with underreporting in lung cancer. Regional variations in testing and reporting were observed., Conclusion: Our study highlights substantial progress in KRAS and BRAF testing in Denmark from 2010 to 2022, evidenced by increased and more specific reporting of mutational test results, thereby improving the precision of cancer diagnosis and treatment. However, persistent regional variations and limited testing for cancer types beyond melanoma, colorectal, and lung cancer highlight the necessity for a nationwide assessment of the optimal testing approach., (© 2024 The Authors. Genes, Chromosomes and Cancer published by Wiley Periodicals LLC.)

Chemical modifications to proteins have wide applications. They may be used in, for example, the production of biopharmaceuticals and fluorescent probes. Despite their importance, highly regioselective chemical protein modifications are often challenging to achieve. We have developed two highly selective methods for protein acylation using poly-His tags inserted either at the N-terminus or in combination with a specific Lys residue. For this, we used an N-terminal Gly-His 6 (Gly-His tag) or the sequence His m -Lys-His n (Lys-His tag), respectively. The Gly-His tag directed the acylation to the N-terminal N α -amine when reacted with 4-methoxyphenyl esters to yield stable conjugates. Next, the Lys-His tag was developed to allow modifications at the C-terminus or in loop regions of proteins. This gave a high selectivity of acylation of the designated Lys N ε -amine in the tag over native Lys residues in the protein under mild conditions. Here, we describe the synthesis of aromatic esters carrying different functionalities and reactivity tuning substituents on the phenol. The expression of poly-His tagged proteins, and the procedure for the highly selective peptide and protein acylations are detailed in this contribution. The versatility of these methods has been demonstrated by the attachment of different functionalities such as fluorophores, biotin, and azides to different proteins and an antibody., (Copyright © 2024. Published by Elsevier Inc.)

Parkinson's disease (PD) may be misdiagnosed due to the clinical overlap between PD and atypical parkinsonism. The utility of α-Synuclein (αSyn) Seed Amplification Assay (SAA) as a diagnostic indicator for PD has been reported in numerous studies, but never when administered as a validated clinical laboratory test. This study compares results from αSyn-SAA validation testing performed using well-characterized cohorts from two biorepositories to better understand the accuracy of PD clinical diagnosis. Blinded cerebrospinal fluid (CSF) specimens from a repository that included cohorts of subjects clinically diagnosed as PD or healthy controls, both with confirmatory dopamine transporter single-photon emission computed tomography (DAT SPECT) imaging, and blinded CSF specimens from a repository that included cohorts of subjects clinically diagnosed as PD or healthy controls based on clinical diagnosis alone, were tested as part of the validation studies for the diagnostic αSyn-SAA test (SYNTap® Biomarker Test). Measured αSyn-SAA test accuracy was 83.9% using clinical diagnosis as comparator, and 93.6% using clinical diagnosis with confirmatory DAT- SPECT imaging as comparator. The statistically significant discordance between accuracy determinations using specimens classified using different diagnostic inclusion criteria indicates that there is some symbiosis between dopamine-weighted imaging and αSyn-SAA results, both of which are associated with higher accuracy compared with the clinical diagnosis alone., (© 2023. The Author(s).)

Background: Quantitative polymerase chain reaction (qPCR) for Epstein-Barr virus (EBV)-DNA is an important diagnostic tool for EBV-associated disease, but interpretation of its clinical significance is challenging., Objectives: We assessed the diagnostic and clinical performance of WHO-standardised qPCR for EBV-DNA (WHO EBV-qPCR) in plasma and whole blood (WB) for proven EBV disease in a prospectively accrued patient cohort., Study Design: Central Denmark Region patients, tested with WHO EBV-qPCR from November 2017 to March 2019, were screened for EBV disease. Incidence (IR) was estimated by Poisson regression. Sensitivity, specificity, positive and negative predictive values (PPV, NPV) were calculated for EBV-qPCR in plasma and WB. Risk of diagnostic latency was compared between patients with EBV-positive and EBV-negative lymphomas., Results: EBV disease was diagnosed in 95 of 1484 participants (IR: 16.3 per 1000 patientyears 95%CI; 13.3-19.9). Sensitivity and specificity of WHO EBV-qPCR in plasma was 82.4% (95% CI; 74.2-90.7%) and 87.8% (95% CI; 85.6-90%), yielding a PPV of 32.2% (95% CI; 24.9-39.5%) and NPV of 98.6% (95% CI; 97.7-99.5%) for proven EBV disease. Sensitivity and NPV were comparable in WB, while specificity and PPV decreased to 66.9% (95% CI; 60.6-73.1%) and 18.1% (95% CI; 7.5-28.7%). Risk of diagnostic latency was 2.3-fold (95% CI 1.4-4.1) higher for patients with EBV-positive compared with EBV-negative lymphomas., Conclusions: WHO EBV-qPCR in plasma and WB have a low PPV but a high NPV for proven EBV disease. Implementation of WHO EBV-qPCR could improve interpretation and facilitate EBV-positive lymphoma diagnosis., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: LUPL and BJ have received an unrestricted grant from Roche Diagnostics. BMB and FdA participate on data safety monitoring boards for the OptimalTTF-2 trial and the Nordic POLAR BEAR trial. FdA is a member of the scientific Committee of the European Society of Haematology, serves as chairman of the ESMO-EHA European Guidelines on Peripheral T-cell Lymphomas and has received grants from the Karen Elise Jensen Foundation, Danish Comprehensive Cancer Center, EU-Horizon, EU-Harmony and Servier. All other authors report no conflict of interest., (Copyright © 2023. Published by Elsevier B.V.)

DNA-stabilized silver nanoclusters (DNA-AgNCs) are biocompatible emitters with intriguing properties. However, they have not been extensively used for bioimaging applications due to the lack of structural information and hence predictable conjugation strategies. Here, a copper-free click chemistry method for linking a well-characterized DNA-AgNC to molecules of interest is presented. Three different peptides and a small protein, human insulin, were tested as labeling targets. The conjugation to the target compounds was verified by MS, HPLC, and time-resolved anisotropy measurements. Moreover, the spectroscopic properties of DNA-AgNCs were found to be unaffected by the linking reactions. For DNA-AgNC-conjugated human insulin, fluorescence imaging studies were performed on Chinese hamster ovary (CHO) cells overexpressing human insulin receptor B (hIR-B). The specific staining of the CHO cell membranes demonstrates that DNA-AgNCs are great candidates for bioimaging applications, and the proposed linking strategy is easy to implement when the DNA-AgNC structure is known.

Background: Clinical and immunological studies in humans show that the live attenuated Bacillus Calmette-Guérin (BCG) vaccine has beneficial non-specific effects, increasing resistance against diseases other than tuberculosis. The underlying mechanisms are currently being explored. The pig exhibits considerable physiological similarity to humans in anatomy and physiology, suggesting that similar responses to BCG could be expected. Studies of the non-specific effects of BCG in pigs are scarce. We investigated the feasibility of using pigs as a large animal model to investigate the non-specific immunological effects of BCG., Methods: In a series of experiments, we randomized newborn or young piglets from conventional farms to receiving BCG or placebo and investigated the persistence of live BCG bacteria in various tissues, the immunogenicity of BCG in ex vivo blood and in vitro stimulation assays, and the acute phase protein and clinical responses to heterologous infectious challenge with influenza A virus or Actinobacillus pleuropneumoniae ., Results: The BCG vaccine was generally well tolerated. In contrast to humans, no skin reaction in the form of abscesses, ulcers, or scars was observed. Live BCG was recovered from draining lymph nodes in 2/13 animals 20 weeks after vaccination. Specific in vitro responses of IFN-γ to antigen-specific re-stimulation with mycobacterial antigen were increased but not TNF-responses to TLR2 or TLR4 agonists. A few genes were differentially expressed in blood after vaccination, including the antiviral genes RIG-I and CSF1 , although the effect disappeared after correction for multiple testing. Clinical symptoms after heterologous bacterial or viral respiratory infections did not differ, nor did virus copies in nasopharyngeal samples after the challenge. However, the acute phase protein response was significantly reduced in BCG-vaccinated animals after influenza challenge but not after A. pleuropneumoniae challenge., Discussion: BCG was safe in pigs, inducing specific immunological responses, but our model did not corroborate the innate immunological responsiveness to BCG seen in humans. The dose of BCG or the bacterial and viral challenges may have been sub-optimal. Even so, the acute phase protein response to influenza infection was significantly reduced in BCG-vaccinated animals., 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 © 2023 Jensen, Hansen, Skovgaard, Svensson, Larsen, Heegaard, Benn and Jungersen.)

Performance in short-duration sports is highly dependent on muscle glycogen, but the total degradation is only moderate and considering the water-binding property of glycogen, unnecessary storing of glycogen may cause an unfavorable increase in body mass. To investigate this, we determined the effect of manipulating dietary carbohydrates (CHO) on muscle glycogen content, body mass, and short-term exercise performance. In a randomized and counterbalanced cross-over design, twenty-two men completed two maximal cycle tests of either 1-min (n = 10) or 15-min (n = 12) duration with different pre-exercise muscle glycogen levels. Glycogen manipulation was initiated three days prior to the tests by exercise-induced glycogen depletion followed by ingestion of a moderate (M-CHO) or high (H-CHO) CHO-diet. Subjects were weighed before each test, and muscle glycogen content was determined in biopsies from m. vastus lateralis before and after each test. Pre-exercise muscle glycogen content was lower following M-CHO than H-CHO (367 mmol · kg -1 DW vs. 525 mmol · kg -1 DW, p < 0.00001), accompanied by a 0.7 kg lower body mass (p < 0.00001). No differences were observed in performance between diets in neither the 1-min (p = 0.33) nor the 15-min (p = 0.99) test. In conclusion, pre-exercise muscle glycogen content and body mass were lower after ingesting moderate compared with high amounts of CHO, while short-term exercise performance was unaffected. This demonstrates that adjusting pre-exercise glycogen levels to the requirements of competition may provide an attractive weight management strategy in weight-bearing sports, particularly in athletes with high resting glycogen levels., (© 2023 The Authors. Scandinavian Journal of Medicine & Science In Sports published by John Wiley & Sons Ltd.)

Background: Knowledge about PFAS exposure in Africa is limited. We have previously detected six types of PFAS in the serum of infants from Guinea-Bissau, West Africa. The aim of this study was to identify predictors of the infant serum-PFAS concentrations., Methods: This cross-sectional study was based on a subset of data from a randomized controlled trial of early measles vaccination performed in 2012-2015 in three rural regions of Guinea-Bissau. Blood samples were obtained from 237 children aged 4-to-7 months, and six types of PFAS were quantified in serum. Location of residence was recorded, and information about predictors related to socioeconomic status as well as maternal and child characteristics were obtained through structured interviews with the mothers through routine surveillance. Associations between potential predictors and infant serum-PFAS concentrations were examined in linear regression models while adjusting for potential confounding and mediating factors as identified in a directed acyclic graph., Results: Infants from the Cacheu region had the lowest concentrations of perfluorooctanoic acid (PFOA), while infants from the Oio region had the lowest concentrations of all other PFAS. Compared to infants from Oio, infant serum-perfluorooctane sulfonic acid (PFOS) concentrations were 94.1% (95% CI: 52.4, 147.1%) and 81.9% (95% CI: 45.7, 127.1%) higher in Cacheu and Biombo, respectively. Higher maternal age and lower parity were associated with slightly higher child-serum perfluorohexane sulfonic acid (PFHxS) concentrations, while infants with higher socioeconomic status and infants breastfed without supplementary solid foods at inclusion had higher average concentrations of most PFAS, although the confidence intervals were wide and overlapped zero., Discussion: Location of residence was the most important determinant of serum-PFAS concentrations among Guinea-Bissau infants, indicating a potential role of diet as affected by the global spread of PFAS, but future studies should explore reasons for the regional differences in PFAS exposure., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Amalie Timmermann reports financial support was provided by Danish Health Foundation (Helsefonden). Ane B Fisker reports financial support was provided by OPTIMUNISE. Philippe Grandjean reports a relationship with Legal cases involving PFAS exposed populations that includes: paid expert testimony., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Objective: The aim of the study was to characterize the drug utilization and switch patterns of biological treatment of ulcerative colitis (UC) and Crohn's disease (CD)., Methods: Using Danish national registries, this nationwide study included individuals diagnosed with UC or CD, bio-naïve at the initiation of treatment with infliximab, adalimumab, vedolizumab, golimumab, or ustekinumab in 2015-2020. Hazard ratios of discontinuing the first treatment or switching to another biological treatment were explored using Cox regression., Results: Among 2995 UC patients and 3028 CD patients, infliximab was used as a first-line biologic treatment in 89% of UC patients and 85% of CD patients, followed by adalimumab with 6%, vedolizumab with 3%, and golimumab with 1% for UC, and adalimumab with 12%, vedolizumab with 2%, and ustekinumab with 0.4% for CD.When comparing adalimumab as the first treatment series to infliximab, there was a higher risk of treatment discontinuation (excluding switch) among UC patients (hazard ratio: 2.02 [95% confidence interval: 1.57; 2.60]) and CD patients (1.85 [1.52; 2.24]). When comparing vedolizumab to infliximab, there was a lower risk of discontinuation for UC patients (0.51 [0.29-0.89]), and for CD patients, although not significantly (0.58 [0.32-1.03]). We observed no significant difference in the risk of switching to another biologic treatment for any of the biologics., Conclusion: More than 85% of UC and CD patients initiating biologic therapy had infliximab as their first-line biologic treatment, in accordance with official treatment guidelines. Future studies should explore the higher incidence of treatment discontinuation of adalimumab as the first treatment series.Key summarySeveral biologic therapies are available in the treatment of ulcerative colitis and Crohn's disease.Clinical guidelines stipulate that infliximab should be the first-line biologic therapy.Drug utilization studies comparing biologic therapies head-to-head are sparse.In Denmark, during 2015-2020 infliximab remained the most widely used biologic treatment, with adalimumab being second.One in four patients experienced more than one biologic during the study period.The risk of discontinuation of biologic treatment (and not starting a new biologic) was higher for initiators of adalimumab.Clinical and social background factors available from the registers could not account for the observed risk difference in discontinuation.

Introduction: Major depressive disorder (MDD) is associated with suicide events and with increased healthcare resource utilization (HRU). The aim was to analyze the pattern of HRU prior to death by suicide or suicide attempt in patients with MDD using national registries., Methods: Danish adults with MDD, who died by suicide or had a first-time suicide attempt, were matched with MDD controls on age, sex, and MDD severity and analyzed for psychiatric and non-psychiatric hospital and private practitioner contacts, and prescriptions 1 year prior to the event. For individuals having a second suicide attempt, HRU prior to first and second suicide attempt was analyzed., Results: Among 1061 individuals dying by suicide and 3759 individuals with suicide attempt, compared with their controls, the proportion with psychiatric hospitalization was more than 50% increased, mainly accounted for by acute contacts. The difference to the matched controls decreased with increasing MDD severity. Non-psychiatric HRU was increased as well. The proportion with psychiatric hospitalizations or ED visits was reduced prior to the second attempt compared with first attempt., Conclusion: Among individuals with MDD, psychiatric and non-psychiatric HRU was increased 1 year prior to suicide event. The proportion of individuals who had psychiatric HRU decreased from first to second suicide attempt., (© 2023 The Authors. Suicide and Life-Threatening Behavior published by Wiley Periodicals LLC on behalf of American Association of Suicidology.)

Genetic variation is a key factor influencing cytokine production capacity, but which genetic loci regulate cytokine production before and after vaccination, particularly in African population is unknown. Here, we aimed to identify single-nucleotide polymorphisms (SNPs) controlling cytokine responses after microbial stimulation in infants of West-African ancestry, comprising of low-birth-weight neonates randomized to bacillus Calmette-Guérin (BCG) vaccine-at-birth or to the usual delayed BCG. Genome-wide cytokine cytokine quantitative trait loci (cQTL) mapping revealed 12 independent loci, of which the LINC01082-LINC00917 locus influenced more than half of the cytokine-stimulation pairs assessed. Furthermore, nine distinct cQTLs were found among infants randomized to BCG. Functional validation confirmed that several complement genes affect cytokine response after BCG vaccination. We observed a limited overlap of common cQTLs between the West-African infants and cohorts of Western European individuals. These data reveal strong population-specific genetic effects on cytokine production and may indicate new opportunities for therapeutic intervention and vaccine development in African populations., Competing Interests: Conflict of interest The authors have no competing interests to declare., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Objectives: We aimed to characterize the advanced NSCLC population in terms of KRAS G12C prevalence, patient characteristics, and survival outcomes after the introduction of immunotherapies., Materials and Methods: We identified adult patients diagnosed with advanced NSCLC between January 1, 2018 and June 30, 2021 using the Danish health registries. Patients were grouped by mutational status (any KRAS mutation, KRAS G12C, and KRAS/EGFR/ALK wildtype [Triple WT]). We analyzed KRAS G12C prevalence, patient and tumor characteristics, treatment history, time-to-next-treatment (TTNT), and overall survival (OS)., Results: We identified 7,440 patients of whom 40% (n = 2,969) were KRAS tested prior to the first line of therapy (LOT1). Among the KRAS tested, 11% (n = 328) harbored KRAS G12C. More KRAS G12C patients were women (67%), smokers (86%), had a high (≥50%) level of PD-L1 expression (54%), and more frequently received anti-PD-L1 treatment than any other group. From the date of the mutational test result, OS (7.1-7.3 months) was similar between the groups. OS from LOT1 (14.0 months) and LOT2 (10.8 months), and TTNT from LOT1 (6.9 months) and LOT2 (6.3 months) was numerically longer for the KRAS G12C mutated group compared to any other group. However, from LOT1 and LOT2, the OS and TTNT were comparable when stratifying the groups by PD-L1 expression level. Regardless of the mutational group, OS was markedly longer for patients with high PD-L1 expression., Conclusion: In patients diagnosed with advanced NSCLC after the implementation of anti-PD-1/L1 therapies, the survival in KRAS G12C mutated patients is comparable to patients with any KRAS mutation, Triple WT, and all NSCLC patients., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: The work was funded by Amgen Inc. The funding was administered by Copenhagen Phase IV Unit, which partly paid the salary of Ditte Gotfredsen, Matilde Grupe Frost, Kristoffer Jarlov Jensen, and Mikkel Zöllner Ankarfeldt. Jon Alexander Lykkegaard reports personal fees from Amgen Inc. during the conduct of the study. Anne Mette Skov Sørensen, Erik Jakobsen, Tonny Studsgaard Petersen and Espen Solem Jimenez have nothing to disclose. Nicholas Sroczynski and Karly Louie were employed at Amgen Limited during the conduct of the study., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Chemical modification of peptides and proteins, such as PEGylation and lipidation, creates conjugates with new properties. However, they are typically not dynamic or stimuli-responsive. Self-assembly controlled by a stimulus will allow adjusting properties directly. Here, we report that conjugates of oligogalacturonic acids (OGAs), isolated from plant-derived pectin, are Ca 2+ -responsive. We report the conjugation of OGA to human insulin (HI) to create new glyco-insulins. In addition, we coupled OGA to model peptides. We studied their self-assembly by dynamic light scattering, small-angle X-ray scattering, and circular dichroism, which showed that the self-assembly to form nanostructures depended on the length of the OGA sequence and Zn 2+ and Ca 2+ concentrations. Subcutaneous administration of OGA12-HI with Zn 2+ showed a stable decrease in blood glucose over a longer period of time compared to HI, despite the lower receptor binding affinity.

Insulin formulations with diverse oligomerization states are the hallmark of interventions for the treatment of diabetes. Here using single-molecule recordings we firstly reveal that insulin oligomerization can operate via monomeric additions and secondly quantify the existence, abundance and kinetic characterization of diverse insulin assembly and disassembly pathways involving addition of monomeric, dimeric or tetrameric insulin species. We propose and experimentally validate a model where the insulin self-assembly pathway is rerouted, favoring monomeric or oligomeric assembly, by solution concentration, additives and formulations. Combining our practically complete kinetic characterization with rate simulations, we calculate the abundance of each oligomeric species from nM to mM offering mechanistic insights and the relative abundance of all oligomeric forms at concentrations relevant both for secreted and administrated insulin. These reveal a high abundance of all oligomers and a significant fraction of hexamer resulting in practically halved bioavailable monomer concentration. In addition to providing fundamental new insights, the results and toolbox presented here can be universally applied, contributing to the development of optimal insulin formulations and the deciphering of oligomerization mechanisms for additional proteins., (© 2023. The Author(s).)

Background: Mental health for engineering undergraduates is an urgent topic for engineering educators. Narratives of engineering education requiring suffering may create or exacerbate problematic perceptions around stress and mental health in engineering. This study explored the roles of stress and mental health in engineering culture. We sought to explore: (1) how engineering students describe their experiences related to stress and mental health and (2) norms and expectations engineering students share about stress and mental health. Qualitative interview data were collected from 30 students who had previously responded to a college-wide survey., Results: Codes related to experiences with stress and mental health in engineering were organized in a bioecological systems model and analyzed for emergent themes depicting engineering culture. The study identified three themes related to stress and mental health in engineering culture: (1) engineering workload as a defining stressor, (2) specific barriers that prevent engineering students from seeking help for mental health concerns, and (3) reliance on peers to cope with stress and mental health distress., Conclusions: Our analysis provided insight into how engineering students perceive norms around stress and mental health in engineering and how this impacts help-seeking for mental health challenges. These findings have important implications for developing interventions and positive cultures that support student mental health., Competing Interests: Competing interestsThe authors declare that they have no competing interest related to this study., (© The Author(s) 2023.)

The chemical modification of proteins is of great importance in chemical biology, biotechnology, and for the production of modified biopharmaceuticals, as it enables introduction of fluorophores, biotin, half-life extending moieties, and more. We have developed two methods that use poly-His sequences to direct the highly selective acylation of proteins, either at the N-terminus or at a specific Lys residue. For the former, we used an N-terminal Gly-His 6 segment (Gly-His tag) that directed acylation of the N-terminal N α -amine with 4-methoxyphenyl esters, resulting in stable conjugates. Next, we developed the peptide sequences His n -Lys-His m (Lys-His tags) that direct the acylation of the designated Lys N ϵ -amine under mild conditions and with high selectivity over native Lys residues. Both the Gly-His and Lys-His tags maintain the capacity for immobilized metal ion affinity chromatography. We have demonstrated the robustness of these methods by attaching different moieties such as azides, fluorophores, and biotin to different proteins, including antibodies., (© 2022 Wiley-VCH GmbH.)

Background: Listening programs enable hearing aid (HA) users to change device settings for specific listening situations and thereby personalize their listening experience. However, investigations into real-world use of such listening programs to support clinical decisions and evaluate the success of HA treatment are lacking., Objective: We aimed to investigate the provision of listening programs among a large group of in-market HA users and the context in which the programs are typically used., Methods: First, we analyzed how many and which programs were provided to 32,336 in-market HA users. Second, we explored 332,271 program selections from 1312 selected users to investigate the sound environments in which specific programs were used and whether such environments reflect the listening intent conveyed by the name of the used program. Our analysis was based on real-world longitudinal data logged by smartphone-connected HAs., Results: In our sample, 57.71% (18,663/32,336) of the HA users had programs for specific listening situations, which is a higher proportion than previously reported, most likely because of the inclusion criteria. On the basis of association rule mining, we identified a primary additional listening program, Speech in Noise, which is frequent among users and often provided when other additional programs are also provided. We also identified 2 secondary additional programs (Comfort and Music), which are frequent among users who get ≥3 programs and usually provided in combination with Speech in Noise. In addition, 2 programs (TV and Remote Mic) were related to the use of external accessories and not found to be associated with other programs. On average, users selected Speech in Noise, Comfort, and Music in louder, noisier, and less-modulated (all P<.01) environments compared with the environment in which they selected the default program, General. The difference from the sound environment in which they selected General was significantly larger in the minutes following program selection than in the minutes preceding it., Conclusions: This study provides a deeper insight into the provision of listening programs on a large scale and demonstrates that additional listening programs are used as intended and according to the sound environment conveyed by the program name., (©Alessandro Pasta, Tiberiu-Ioan Szatmari, Jeppe Høy Christensen, Kasper Juul Jensen, Niels Henrik Pontoppidan, Kang Sun, Jakob Eg Larsen. Originally published in the Journal of Medical Internet Research (https://www.jmir.org), 17.10.2022.)

Bile acids are crucial for the uptake of dietary lipids and can shape the gut-microbiome composition. This latter function is associated with the toxicity of bile acids and can be modulated by bile acid modifying bacteria such as Eggerthella lenta , but the molecular details of the interaction of bacteria depending on bile acid modifications are not well understood. In order to unravel the molecular response to bile acids and their metabolites, we cultivated eight strains from a human intestinal microbiome model alone and in co-culture with Eggerthella lenta in the presence of cholic acid (CA) and deoxycholic acid (DCA). We observed growth inhibition of particularly gram-positive strains such as Clostridium ramosum and the gram-variable Anaerostipes cacae by CA and DCA stress. C. ramosum was alleviated through co-culturing with Eggerthella lenta . We approached effects on the membrane by zeta potential and genotoxic and metabolic effects by (meta)proteomic and metabolomic analyses. Co-culturing with Eggerthella lenta decreased both CA and DCA by the formation of oxidized and epimerized bile acids. Eggerthella lenta also produces microbial bile salt conjugates in a co-cultured species-specific manner. This study highlights how the interaction with other bacteria can influence the functionality of bacteria.

Objectives: To investigate healthcare resource utilization (HRU) and associated costs by depression severity and year of diagnosis among patients with treatment-resistant depression (TRD) in Denmark., Methods: Including all adult patients with a first-time hospital contact for major depressive disorder (MDD) in 1996-2015, TRD patients were defined at the second shift in depression treatment (antidepressant medicine or electroconvulsive therapy) and matched 1:2 with non-TRD patients. The risk of utilization and amount of HRU and associated costs including medicine expenses 12 months after the TRD-defining date were reported, comparing TRD patients with non-TRD MDD patients., Results: Identifying 25,321 TRD-patients matched with 50,638 non-TRD patients, the risk of psychiatric hospitalization following TRD diagnosis was 138.4% (95%-confidence interval: 128.3-149.0) higher for TRD patients than for non-TRD MDD patients. The number of hospital bed days and emergency department (ED) visits were also higher among TRD patients, with no significant difference for somatic HRU. Among patients who incurred healthcare costs, the associated HRU costs for TRD patients were 101.9% (97.5-106.4) higher overall, and 55.2% (50.9-59.6) higher for psychiatric services than those of non-TRD patients. The relative differences in costs for TRD-patients vs non-TRD patients were greater for patients with mild depression and tended to increase over the study period (1996-2015), particularly for acute hospitalizations and ED visits., Limitations: TRD was defined by prescription patterns besides ECT treatments., Conclusion: TRD was associated with increased psychiatric-related HRU. Particularly the difference in acute hospitalizations and ED visits between TRD and non-TRD patients increased over the study period., Competing Interests: The study was performed by the Copenhagen Phase IV Unit (Phase4CPH) and was financed by Janssen Cilag A/S, which holds license to antidepressant medicine. Via Phase4CPH, JP, EJS and MZA have performed other studies regarding antidepressants involving funding from Janssen Cilag and Eli Lilly, while KJJ, FHG and MO have performed other studies regarding antidepressants involving funding from Janssen Cilag. All funds were given to their institution. JR, NB and SA are employees of Janssen Cilag. Janssen Cilag A/S and its employees did not have access to the registry data located on the servers of Statistics Denmark and had therefore no influence on the data management and data analysis of the present work. The authors at Copenhagen Phase IV Unit retained editorial control of the manuscript. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Background: Early 2-dose measles vaccine (MV) at 4 and 9 months of age vs. the WHO strategy of MV at 9 months of age reduced all-cause child mortality in a previous trial. We aimed to test two hypotheses: 1) a 2-dose strategy reduces child mortality between 4 and 60 months of age by 30%; 2) receiving early MV at 4 months in the presence versus absence of maternal measles antibodies (MatAb) reduces child mortality by 35%., Methods: Single-centre open-label community-based randomised controlled trial in Guinea-Bissau, with 2:1 block-randomisation by sex to a 2-dose (4 + 9 months) vs. 1-dose (9 months) MV strategy. Healthy children were eligible 4 weeks after the 3rd diphtheria-tetanus-pertussis-containing vaccine. Before randomisation a blood sample was collected to determine MatAb level. The primary outcome was all-cause mortality. Hazard ratios (HR) were derived from Cox regression in the per protocol population. We tested for interactions with national campaigns with oral polio vaccine (C-OPV). Trial registration: NCT01486355., Findings: Between August 2011-April 17th 2015, 6,636 children were enroled, 6,598[n 2-dose =4,397; n 1-dose =2,201] were included in the analysis of the primary outcome, The HR(2-dose/1-dose) between 4 and 60 months was 1.38 (95%CI: 0.92-2.06) [deaths: n 2-dose =90; n 1-dose =33]. Before the 9-month MV and the HR(1-dose/no dose) was 0.94 (0.45-1.96) [deaths: n 2-dose =21; n 1-dose =11]. The HR(2-dose/1-dose) was 0.81 (0.29-2.22) for children, who received no C-OPV [deaths/children: n 2-dose =10/2,801; n 1-dose =6/1,365], and 4.73 (1.44-15.6) for children, who received C-OPV before and after enrolment (p for interaction=0.027) [deaths/children: n 2-dose =27/1,602; n 1-dose =3/837]. In the 2-dose group receiving early MV at 4 months, mortality was 50% (20-68%) lower for those vaccinated in the presence of MatAb vs. the absence of MatAb [deaths/children: n MatAb =51/3,132; n noMatAb =31/1,028]., Interpretation: The main result contrasts with previous findings but may, though based on a small number of events, be explained by frequent OPV campaigns that reduced the mortality rate, but apparently interacted negatively with early MV. The beneficial non-specific effects of MV in the presence of MatAb should be investigated further., Funding: ERC, Danish National Research Foundation, the Danish Council for Development Research, Ministry of Foreign Affairs, Novo Nordisk Foundation, European Union and the Lundbeck Foundation., Competing Interests: OB received a scholarship from the Lundbeck Foundation. All other authors declare no competing interests., (© 2022 The Authors.)

Vaccines have generally been developed with limited insight into their molecular impact. While systems vaccinology enables characterization of mechanisms of action, these tools have yet to be applied to infants, who are at high risk of infection and receive the most vaccines. Bacille Calmette-Guérin (BCG) protects infants against disseminated tuberculosis (TB) and TB-unrelated infections via incompletely understood mechanisms. We employ mass-spectrometry-based metabolomics of blood plasma to profile BCG-induced infant responses in Guinea-Bissau in vivo and the US in vitro. BCG-induced lysophosphatidylcholines (LPCs) correlate with both TLR-agonist- and purified protein derivative (PPD, mycobacterial antigen)-induced blood cytokine production in vitro, raising the possibility that LPCs contribute to BCG immunogenicity. Analysis of an independent newborn cohort from The Gambia demonstrates shared vaccine-induced metabolites, such as phospholipids and sphingolipids. BCG-induced changes to the plasma lipidome and LPCs may contribute to its immunogenicity and inform the development of early life vaccines., Competing Interests: Declaration of interests O.L. is a named inventor on several Boston Children’s Hospital patents relating to human microphysiologic assay systems and vaccine adjuvants. S.M. and G.M. are employees of Metabolon. The other authors declare no competing financial interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Somatostatin and its related peptides (SSRPs) form an important family of hormones with diverse physiological roles. The ubiquitous presence of SSRPs in vertebrates and several invertebrate deuterostomes suggests an ancient origin of the SSRP signaling system. However, the existence of SSRP genes outside of deuterostomes has not been established, and the evolutionary history of this signaling system remains poorly understood. Our recent discovery of SSRP-like toxins (consomatins) in venomous marine cone snails (Conus) suggested the presence of a related signaling system in mollusks and potentially other protostomes. Here, we identify the molluscan SSRP-like signaling gene that gave rise to the consomatin family. Following recruitment into venom, consomatin genes experienced strong positive selection and repeated gene duplications resulting in the formation of a hyperdiverse family of venom peptides. Intriguingly, the largest number of consomatins was found in worm-hunting species (>400 sequences), indicating a homologous system in annelids, another large protostome phylum. Consistent with this, comprehensive sequence mining enabled the identification of SSRP-like sequences (and their corresponding orphan receptor) in annelids and several other protostome phyla. These results established the existence of SSRP-like peptides in many major branches of bilaterians and challenge the prevailing hypothesis that deuterostome SSRPs and protostome allatostatin-C are orthologous peptide families. Finally, having a large set of predator-prey SSRP sequences available, we show that although the cone snail's signaling SSRP-like genes are under purifying selection, the venom consomatin genes experience rapid directional selection to target receptors in a changing mix of prey., (© The Author(s) 2022. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

Chemical modification of proteins has numerous applications, but it has been challenging to achieve the required high degree of selectivity on lysine amino groups. Recently, we described the highly selective acylation of proteins with an N-terminal Gly-His 6 segment. This tag promoted acylation of the N-terminal N α -amine resulting in stable conjugates. Herein, we report the peptide sequences His n -Lys-His m , which we term Lys-His tags. In combination with simple acylating agents, they facilitate the acylation of the designated Lys N ϵ -amine under mild conditions and with high selectivity over native Lys residues. We show that the Lys-His tags, which are 7 to 10 amino acids in length and still act as conventional His tags, can be inserted in proteins at the C-terminus or in loops, thus providing high flexibility regarding the site of modification. Finally, the selective and efficient acylation of the therapeutic antibody Rituximab, pure or mixed with other proteins, demonstrates the scope of the Lys-His tag acylation method., (© 2022 Wiley-VCH GmbH.)

Introduction: Human non-live vaccines have been associated with detrimental non-specific effects (NSE), particularly in females. A large trial found 2-fold increased overall mortality in girls receiving a new malaria vaccine compared to the rabies vaccine used as a coontrol; a beneficial NSE of the rabies vaccine was proposed. Conversely, in dogs increased mortality was seen in females but not males following rabies vaccination of puppies born to immunized mothers. We investigated NSE of non-live rabies vaccine in piglets and the potential modifying effect of maternal priming with rabies vaccine., Methods: In a Danish herd of commercial rabies virus-free pigs, 575 pregnant sows (2-3 weeks before scheduled farrowing) and 5747 of their offspring (median 6-day-old) were allocated (1:1) to non-live rabies vaccine (Versiguard rabies vet) or no rabies vaccine. Outcomes were overall mortality and antibiotic treatment until departure from the nursery (approximately age 12 weeks/30 kgs)., Results: Until weaning, overall offspring mortality was 2.2% (127 piglets died, rabies vaccine: n = 69; control: n = 58), the proportion ratio (PR) being 1.19 (95% confidence interval: 0.84-1.68). Until end of follow-up, mortality was 4.1% (233, rabies vaccine: n = 115; control = 118, PR: 0.97 (0.76-1.25)). Prior sow rabies vaccination did not affect piglet mortality. For mortality as well as risk of antibiotic treatment before weaning, there was indication of a beneficial effect of rabies vaccine in female piglets, but a negative effect in (castrated) male piglets from rabies-naïve sows. Prior sow vaccination significantly modified the vaccine effect estimate in female piglets toward a detrimental effect of rabies vaccine on treatment risk. These effects had waned by 12 weeks of age., Conclusion: The study did not support the hypothesized beneficial NSE of rabies vaccine. Although under-powered for subgroup analyses, the study indicated effect modification by sex and maternal vaccination. Results could be different in a herd with higher mortality and infectious burden., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021. Published by Elsevier Ltd.)

Human insulin (HI) has fascinating metal-facilitated self-assembly properties that are essential for its biological function. HI has a natural Zn 2+ binding site and we have previously shown that covalently attached abiotic ligands (e.g., bipyridine, terpyridine) can lead to the formation of nanosized oligomeric structures through the coordination of metal ions. Here we studied the hypothesis that metal ions can be used to directly control the pharmacokinetics of insulin after covalent attachment of an abiotic ligand that binds metal ions. We evaluated the pharmacokinetics (PK) and biodistribution of HI self-assemblies directed by metal ion coordination (i.e., Fe 2+ /Zn 2+ , Eu 3+ /Zn 2+ , Fe 2+ /Co 3+ ) using preclinical SPECT/CT imaging and ex vivo gamma counting. HI was site-specifically modified with terpyridine (Tpy) at the Phe B1 or Lys B29 position to create conjugates that bind either Fe 2+ or Eu 3+ , while its natural binding site (His B10 ) preferentially coordinates with either Zn 2+ or Co 3+ . HI was also functionalized with trans-cyclooctene (TCO) opposite to Tpy at Phe B1 or Lys B29 , respectively, to allow for tetrazine-TCO coupling via a tetrazine-modified DTPA followed by 111 In-radiolabeling for SPECT/CT imaging. When the 111 In-B29Tpy-HI conjugate was coordinated with Fe 2+ /Zn 2+ , its retention at the injection site 6 h after injection was ~8-fold higher than the control without the metal ions, while its kidney accumulation was lower. 111 In-B1Tpy-HI showed comparable retention at the injection site 6 h after injection and slightly increased retention at 24 h. However, higher kidney accumulation and residence time of degraded 111 In-B1Tpy-HI was observed compared to that of 111 In-B29Tpy-HI. Quantitative PK analysis based on SPECT/CT images confirmed slower distribution from the injection site of the HI-metal ion assemblies compared to control HI conjugates. Our results show that the Tpy-binding site (i.e., Phe B1 or Lys B29 ) on HI and its coordination with the added metal ions (i.e., Fe 2+ /Zn 2+ or Fe 2+ /Co 3+ ) directed the distribution half-life of HI significantly. This clearly indicates that the PK of insulin can be controlled by complexation with different metal ions., (Copyright © 2022. Published by Elsevier B.V.)

Chiral communications exist in secondary structures of foldamers and copolymers via a network of noncovalent interactions within effective intermolecular force (IMF) range. It is not known whether long-range chiral communication exists between macromolecular tertiary structures such as peptide coiled-coils beyond the IMF distance. Harnessing the high sensitivity of single-molecule force spectroscopy, we investigate the chiral interaction between covalently linked DNA duplexes and peptide coiled-coils by evaluating the binding of a diastereomeric pair of three DNA-peptide conjugates. We find that right-handed DNA triple helices well accommodate peptide triple coiled-coils of the same handedness, but not with the left-handed coiled-coil stereoisomers. This chiral communication is effective in a range (<4.5 nm) far beyond canonical IMF distance. Small-angle X-ray scattering and molecular dynamics simulation indicate that the interdomain linkers are tightly packed via hydrophobic interactions, which likely sustains the chirality transmission between DNA and peptide domains. Our findings establish that long-range chiral transmission occurs in tertiary macromolecular domains, explaining the presence of homochiral pairing of superhelices in proteins., (© 2022. The Author(s).)

Mental health challenges have been rising across college campuses. To destigmatize wellness practices and promote student mental health, we present a novel technical project in an introductory bioengineering course that explores stress management techniques through physiology, biosensors, and design. We hypothesize that if students measure objective, physiologic impacts of stress management techniques on themselves, they may be more likely to realize the benefits and use those techniques when needed. Additionally, through this data-driven project, we aim to appeal to engineers' critical thinking nature. To support students in selecting stress management techniques for themselves, mindfulness is introduced and practiced in the course. Initial student feedback on the introduction of mindfulness into the classroom is positive. The COVID-19 pandemic has emphasized the need to focus on student wellbeing in addition to physical health. Integration of wellness into the core curriculum can normalize the use of these resources within engineering departments and colleges and equip students with stress management tools for their careers. Ultimately, this curricular development lays the groundwork for institutional enhancement of undergraduate STEM education by supporting student wellness through the engineering curriculum., Supplementary Information: The online version contains supplementary material available at 10.1007/s43683-021-00060-1., (© The Author(s), under exclusive licence to Biomedical Engineering Society 2021.)

The use of C-terminal peptide thioesters and hydrazides in synthetic protein chemistry has inspired the search for optimal solid-phase peptide synthesis (SPPS) strategies for their assembly. However, peptide thioesters are not directly accessible by conventional Fmoc-SPPS owing to the nucleophilicity of the secondary amine required for Fmoc removal. Here, we report the mild and effective activation of the pGlu linker and a new safety-catch linker that was used for the convenient synthesis of peptide thioesters and hydrazides via efficient amide-to-imide activation followed by nucleophilic displacement., (© 2021 European Peptide Society and John Wiley & Sons, Ltd.)

This study investigated the postprandial plasma metabolome following consumption of four dairy matrices different in texture and structure: cheddar cheese (Cheese), homogenized cheddar cheese (Hom. Cheese), and micellar casein isolate (MCI) with cream (MCI Drink) or a MCI Gel. An acute, randomized, crossover trial in male participants (n = 25) with four test days was conducted. Blood samples were collected during an 8-h postprandial period after consumption of a meal similar in micro- and macronutrients containing one of the four dairy matrices, and the metabolome was analyzed using nuclear magnetic resonance (NMR) spectroscopy. A liquid dairy matrix (MCI Drink) resulted in a faster absorption of amino acids compared to products, representing either a semi-solid (MCI Gel and Hom. Cheese) or solid (Cheese) dairy matrix. For the MCI Gel, plasma concentration of acetic acid and formic acid increased approximately 2 h following consumption, while 3-hydroxybyturate and acetoacetic acid increased approximately 6 h after consumption. The structure and texture of the dairy matrix affected the postprandial absorption of amino acids, as revealed by the plasma metabolome. Our study furthermore pointed at endogenous effects associated with consumption of dairy products containing glucono-δ-lactone.

Background: The dairy matrix may influence digestion and absorption of lipids and thereby risk of cardiovascular diseases (CVDs). However, few postprandial studies have compared dairy products that differed only in terms of their matrix., Objectives: We aimed to investigate acute 8-h postprandial lipid, glycemic, and appetite responses after intake of isoenergetic dairy meals with different matrixes, but similar nutritional composition., Methods: Twenty-five normal-weight men (18-40 y old) were enrolled in a randomized controlled crossover trial. On 4 test days, a meal with 1 of 4 dairy products was served: cheddar cheese (Cheese), homogenized Cheese (Hom. Cheese), micellar casein isolate (MCI) with cream (MCI Drink), and a gel produced from the MCI Drink by addition of Glucono Delta-Lactone (MCI Gel). The fat- and protein-matched dairy products differed in terms of their casein network, fat droplet size, and/or texture. Blood biochemistry and appetite responses were collected., Results: Eighteen participants completed the trial. Postprandial triglycerides (TGs) (primary outcome) increased by (mean ± SEM) 0.24 ± 0.07 and 0.19 ± 0.07 mmol/L after MCI Gel compared with Cheese and Hom. Cheese, respectively (both P ≤ 0.05). Likewise, MCI Gel increased TG incremental AUC compared with Cheese and Hom. Cheese (both P < 0.05), and peak compared with Cheese (P < 0.05). ApoB-48 (primary outcome) was unaffected by dairy matrix. For free fatty acids (FFAs), glucose, and insulin, time × meal interactions were observed (all P < 0.001). During the first 2 h, FFAs were lower for Cheese than for MCI products, whereas the opposite was observed for glucose and insulin., Conclusions: Postprandial TG but not apoB-48 response was higher after MCI Gel, indicating that the type of casein network influences lipid responses. This suggests that the dairy matrix may also affect risk factors for CVDs. Reducing fat droplet size (i.e., Hom. Cheese) did not affect blood biochemistry.This trial was registered at clinicaltrials.gov as NCT03656367., (© The Author(s) 2021. Published by Oxford University Press on behalf of the American Society for Nutrition.)

Plants and animals use cell surface receptors to sense and interpret environmental signals. In legume symbiosis with nitrogen-fixing bacteria, the specific recognition of bacterial lipochitooligosaccharide (LCO) signals by single-pass transmembrane receptor kinases determines compatibility. Here, we determine the structural basis for LCO perception from the crystal structures of two lysin motif receptor ectodomains and identify a hydrophobic patch in the binding site essential for LCO recognition and symbiotic function. We show that the receptor monitors the composition of the amphiphilic LCO molecules and uses kinetic proofreading to control receptor activation and signaling specificity. We demonstrate engineering of the LCO binding site to fine-tune ligand selectivity and correct binding kinetics required for activation of symbiotic signaling in plants. Finally, the hydrophobic patch is found to be a conserved structural signature in this class of LCO receptors across legumes that can be used for in silico predictions. Our results provide insights into the mechanism of cell-surface receptor activation by kinetic proofreading of ligands and highlight the potential in receptor engineering to capture benefits in plant-microbe interactions., Competing Interests: Competing interest statement: K.G., Z.B., L.H.M., S.R., J.S., and K.R.A., are inventors on patent application 62/718,186 submitted by Aarhus University that covers LysM receptors: genetically altered LysM receptors with altered agonist specificity and affinity. M.L., D.L., S.R., J.S., and K.R.A., are inventors on patent application 63/027,151 submitted by Aarhus University that covers LysM receptor motifs., (Copyright © 2021 the Author(s). Published by PNAS.)

In obesity, excessive abdominal fat, especially the accumulation of visceral adipose tissue (VAT), increases the risk of metabolic disorders, such as type 2 diabetes mellitus (T2DM), cardiovascular disease, and non-alcoholic fatty liver disease. Excessive abdominal fat is associated with adipose tissue dysfunction, leading to systemic low-grade inflammation, fat overflow, ectopic lipid deposition, and reduced insulin sensitivity. Physical activity is recommended for primary prevention and treatment of obesity, T2DM, and related disorders. Achieving a stable reduction in body weight with exercise training alone has not shown promising effects on a population level. Because fat has a high energy content, a large amount of exercise training is required to achieve weight loss. However, even when there is no weight loss, exercise training is an effective method of improving body composition (increased muscle mass and reduced fat) as well as increasing insulin sensitivity and cardiorespiratory fitness. Compared with traditional low-to-moderate-intensity continuous endurance training, high-intensity interval training (HIIT) and sprint interval training (SIT) are more time-efficient as exercise regimens and produce comparable results in reducing total fat mass, as well as improving cardiorespiratory fitness and insulin sensitivity. During high-intensity exercise, carbohydrates are the main source of energy, whereas, with low-intensity exercise, fat becomes the predominant energy source. These observations imply that HIIT and SIT can reduce fat mass during bouts of exercise despite being associated with lower levels of fat oxidation. In this review, we explore the effects of different types of exercise training on energy expenditure and substrate oxidation during physical activity, and discuss the potential effects of exercise training on adipose tissue function and body fat distribution., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Kolnes, Petersen, Lien-Iversen, Højlund and Jensen.)

Plant metabolism depends on cascade reactions mediated by dynamic enzyme assemblies known as metabolons. In this context, the cytochrome P450 (P450) superfamily catalyze key reactions underpinning the unique diversity of bioactive compounds. In contrast to their soluble bacterial counterparts, eukaryotic P450s are anchored to the endoplasmic reticulum membrane and serve as metabolon nucleation sites. Hence, membrane anchoring appears to play a pivotal role in the evolution of complex biosynthetic pathways. Here, a model membrane assay enabled characterization of membrane anchor dynamics by single molecule microscopy. As a model system, we reconstituted the membrane anchor of cytochrome P450 oxidoreductase (POR), the ubiquitous electron donor to all microsomal P450s. The transmembrane segment in the membrane anchor of POR is relatively conserved, corroborating its functional importance. We observe dynamic colocalization of the POR anchors in our assay suggesting that membrane anchoring might promote intermolecular interactions and in this way impact assembly of metabolic multienzyme complexes., (© 2021. The Author(s).)

While the assessment of hearing aid use has traditionally relied on subjective self-reported measures, smartphone-connected hearing aids enable objective data logging from a large number of users. Objective data logging allows to overcome the inaccuracy of self-reported measures. Moreover, data logging enables assessing hearing aid use with a greater temporal resolution and longitudinally, making it possible to investigate hourly patterns of use and to account for the day-to-day variability. This study aims to explore patterns of hearing aid use throughout the day and assess whether clusters of users with similar use patterns can be identified. We did so by analyzing objective hearing aid use data logged from 15,905 real-world users over a 4-month period. Firstly, we investigated the daily amount of hearing aid use and its within-user and between-user variability. We found that users, on average, used the hearing aids for 10.01 h/day, exhibiting a substantial between-user (SD = 2.76 h) and within-user (SD = 3.88 h) variability. Secondly, we examined hearing aid use hourly patterns by clustering 453,612 logged days into typical days of hearing aid use. We identified three typical days of hearing aid use: full day (44% of days), afternoon (27%), and sporadic evening (26%) day of hearing aid use. Thirdly, we explored the usage patterns of the hearing aid users by clustering the users based on the proportion of time spent in each of the typical days of hearing aid use. We found three distinct user groups, each characterized by a predominant (i.e., experienced ~60% of the time) typical day of hearing aid use. Notably, the largest user group (49%) of users predominantly had full days of hearing aid use. Finally, we validated the user clustering by training a supervised classification ensemble to predict the cluster to which each user belonged. The high accuracy achieved by the supervised classifier ensemble (~86%) indicated valid user clustering and showed that such a classifier can be successfully used to group new hearing aid users in the future. This study provides a deeper insight into the adoption of hearing care treatments and paves the way for more personalized solutions., Competing Interests: AP, T-IS, and KJ are employed by Demant A/S. JC, NP, and KS are employed by Oticon A/S. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Pasta, Szatmari, Christensen, Jensen, Pontoppidan, Sun and Larsen.)

Objective: Patients increasingly seek information on their medical conditions from the internet. The present study evaluated the quality and readability of readily available online patient resources for varicose veins., Methods: An internet search for "varicose veins" was conducted using the meta-search engines Yippy and Dogpile and the general search engines Google, Yahoo, and Bing with a cleared-cache web browser in July 2019. Two trained raters scored the websites separately on the dimensions of accessibility, accountability, interactivity, structure, and content. Any discrepancies were discussed, and a consensus was reached. Readability was calculated using four readability metric systems. Rater consistency was evaluated using kappa, weighted kappa, and interrater correlation coefficient, as indicated., Results: A total of 189 websites met the inclusion criteria. The total median quality score was 15.6 (interquartile range [IQR], 13.1-20.5; range, 7.4-31.3) of 38. The websites scored a median of 4 (IQR, 1-8) of 15 for accountability, 2 (IQR, 2-2) of 5 for interactivity, 4 (IQR, 2-4) of 4 for organization, and 6.4 (IQR, 3.9-7.9) of 14 for weighted content. Most websites (81.5%) were accessible. However, the overall readability was poor. The median Flesch-Kincaid reading ease score was 55.1 (IQR, 49.4-6.7), indicating that the text was fairly difficult to read. The median grade level was 10th grade using both the Flesch-Kincaid grade level and simple measure of the Gobbledygook index and 11th to 12th grade using the new Dale-Chall readability formula. Government websites were the most accountable, featured the best content, and were the most readable. The website traffic had a positive, nonlinear correlation with the total score and a negative, nonlinear correlation with the website rank (or position on the search result page). Website rank correlated negatively with the total score, although the correlation was weak., Conclusions: The quality of the online patient resources on varicose veins varies greatly, and the readability for most sites is poor. Government-sponsored websites had the highest quality and were the most readable. Physicians are advised to consider providing a list of appropriate websites to their patients to better inform them, avoid confusion, and ensure appropriate delivery of accurate and readable information., (Copyright © 2020 Society for Vascular Surgery. Published by Elsevier Inc. All rights reserved.)

Glioblastoma (GBM) is a devastating cancer with basically no curative treatment. Even with aggressive treatment, the median survival is disappointing 14 months. Surgery remains the key treatment and the postoperative survival is determined by the extent of resection. Unfortunately, the invasive growth with irregular infiltrating margins complicates an optimal surgical resection. Precise intraoperative tumor visualization is therefore highly needed and molecular targeted near-infrared (NIR) fluorescence imaging potentially constitutes such a tool. The urokinase-type Plasminogen Activator Receptor (uPAR) is expressed in most solid cancers primarily at the invading front and the adjacent activated peritumoral stroma making it an attractive target for targeted fluorescence imaging. The purpose of this study was to develop and evaluate a new uPAR-targeted optical probe, IRDye800CW-AE344, for fluorescence guided surgery (FGS). Methods: In the present study we characterized the fluorescent probe with regard to binding affinity, optical properties, and plasma stability. Further, in vivo imaging characterization was performed in nude mice with orthotopic human patient derived glioblastoma xenografts, and we performed head-to-head comparison within FGS between our probe and the traditional procedure using 5-ALA. Finally, the blood-brain barrier (BBB) penetration was characterized in a 3D BBB spheroid model. Results: The probe effectively visualized GBM in vivo with a tumor-to-background ratio (TBR) above 4.5 between 1 to 12 h post injection and could be used for FGS of orthotopic human glioblastoma xenografts in mice where it was superior to 5-ALA. The probe showed a favorable safety profile with no evidence of any acute toxicity. Finally, the 3D BBB model showed uptake of the probe into the spheroids indicating that the probe crosses the BBB. Conclusion: IRDye800CW-AE344 is a promising uPAR-targeted optical probe for FGS and a candidate for translation into human use., Competing Interests: Competing Interests: AK is a co-founder and KJ is an employee of FluoGuide A/S that seeks to commercialize uPAR-targeted optical probes., (© The author(s).)

Objective: To describe treatment patterns in patients with treatment-resistant depression (TRD) and major depressive disorder (MDD) stratified by depression severity and year of diagnosis. Patterns of treatment were also compared to country-specific guidelines., Methods: All adults registered first time with a hospital contact due to MDD from 1996 through 2015 were identified and followed for all dispensed prescriptions of antidepressants, antipsychotics, lithium, initiation of electroconvulsive therapy (ECT), and psychotherapy in Danish registers 12 months before and after their hospital MDD diagnosis. TRD was characterized by two shifts in treatment., Results: We identified 197,615 patients of whom 15% developed TRD. In total, 88% of patients started treatment with antidepressants or ECT. Selective serotonin reuptake inhibitors (SSRIs) were the most frequently used treatment during the study period and more than half (50.7%) of patients changed treatment at least once. Among patients with TRD, serotonin and noradrenaline reuptake inhibitors (SNRIs) were the most frequently used treatment (55.9%), and 37.0% initiated a new treatment the following year. SSRIs and SNRIs were part of most combinations of treatment, regardless of depression severity, year of diagnosis, or presence of TRD., Conclusion: 15% of patients met the criteria for TRD. Irrespective of patient characteristics and year of diagnosis, SSRIs and SNRIs are the most used treatments for depression, even after patients met the criteria for TRD. We confirm that guidelines for first treatment were followed for most patients diagnosed with MDD in Denmark, but for patients with TRD, choice of treatment was arbitrary., (Copyright © 2021. Published by Elsevier B.V.)

Metal ion-induced self-assembly (SA) of proteins into higher-order structures can provide new, dynamic nano-assemblies. Here, the synthesis and characterization of a human insulin (HI) analog modified at Lys B29 with the tridentate chelator 2,2':6',2''-terpyridine (Tpy) is described. SA of this new insulin analog (Lys B29 Tpy-HI) in the presence of the metal ions Fe 2+ and Eu 3+ at different concentrations was studied in solution by fluorescence luminescence and CD spectroscopy, dynamic light scattering, and small-angle X-ray scattering, while surface assembly was probed by AFM. Unique oligomerization was observed in solution, as Fe 2+ yielded small magenta-colored discrete non-native assemblies, while Eu 3+ caused the formation of large fractal assemblies. Binding of both metal ions to Tpy was demonstrated spectroscopically, and emission lifetime experiments revealed a distinct Eu 3+ coordination geometry that included two water molecules. SAXS suggested that Lys B29 Tpy-HI with Fe 2+ oligomerized to a discrete, roughly octameric species, while Lys B29 Tpy-HI with Eu 3+ gave very large assemblies that could be modelled as fractals. The fractal dimensionality increased with the Eu 3+ concentration. We propose that this is a consequence of Eu 3+ binding to both Tpy and to free carboxylic acid groups on the insulin surface. Lys B29 Tpy-HI maintained insulin receptor affinity, and showed extended blood glucose lowering and plasma concentration after subcutaneous injection in rats. The combination of metal ion directed SA and native SA provides control of nano-scale fractal dimensionality and points towards use in therapeutics.