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This study explores the feasibility of utilizing in vitro cultivated milk-derived bovine mammary epithelial cells (bMECs) for the production of milk constituents. BMECs were isolated from milk and treated with various lactogenic agents in 3D transwell systems. By proteomics, >900 proteins were identified and quantified in the secretomes, including >100 milk-related proteins such as caseins and enzymes. Despite limited secretion of total proteins and major milk proteins, 110 proteins were found phosphorylated, including 27 involved in metal- or calcium-binding. Mineral analysis confirmed that 6-9 % of minerals in secretomes were associated with proteins. Notably, six proteins, including prolactin, were secreted into the basolateral side of bMECs without lactogenic treatment, suggesting their local de novo synthesis. This research advances our understanding of bMECs biology, as well as the compositional and functional features of their secretomes, highlighting their potential for sustainable production of functional milk proteins, meanwhile emphasizing the need for further optimization., 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 © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

During processing and storage of both conventional and lactose-hydrolyzed UHT milk (LHM), aggregation of milk proteins occurs. Protein aggregation can inter alia occur via nonreducible covalent cross-links derived from either Maillard or dehydroalanine (DHA) pathways. To study this further in relation to processing method and lactase enzyme purity, LHM was produced using 3 different lactase preparations, with lactase enzymes added in a dairy setting either before (prehydrolysis) or after (posthydrolysis) UHT treatment. The prepared LHM types were subsequently stored at either 25°C or 35°C for up to one year. Mass spectrometry was used to absolutely quantify the level of furosine, N-ε-(carboxymethyl)lysine, N-ε-(carboxyethyl)lysine, lanthionine, and lysinoalanine in these products using a newly developed method on triple quadrupole for these processing-induced markers. The results showed higher levels of Maillard-related processing markers in prehydrolyzed LHM compared with posthydrolyzed LHM and conventional UHT milk, which, on the other hand, contained higher concentrations of DHA-derived cross-links. Proteomics of collected particles from asymmetrical flow field-flow fractionation in combination with gel electrophoresis indicated the presence of intramicellar cross-links during storage, especially for larger particles involving α S1 - and α S2 -caseins., (© 2024, The Authors. Published by Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Introduction: In Denmark, there are no official guidelines on how to inform women about the risk of recall during mammography screening, leading to varied local practices. This study explored the experiences of radiographers at a Danish mammography screening unit and breast cancer assessment clinic communicating the risk of recall and false-positive results., Methods: This study involved 12 weeks of ethnographic fieldwork over the course of one year, along with eleven semi-structured interviews with radiographers., Results: Radiographers worked at the screening unit and the breast cancer assessment clinic. This provided them an opportunity to learn from their colleagues, improve communication skills, and the ability to recognise and respond to emotional reactions. The radiographers were aware that recalls might cause psychosocial consequences and had thus established a local practice sensitive to this. The radiographers informed women at their first screening about the risk of recall and false positives, aiming to mitigate these consequences., Conclusion: These findings highlight the advantages of radiographers working in both screening and clinical settings., Implications for Practice: Insights from this study may inspire future guidelines or local screening practices and improve patient care. Furthermore, results may inform department managers' workload organisation., Competing Interests: Conflict of interest statement The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Level of Evidence: Therapeutic Level I . See Instructions for Authors for a complete description of levels of evidence., Competing Interests: Disclosure: Funding for this study was received from Sophies Minde Ortopedi AS, The South-East Health Authority Norway, and The Research Council of Norway. The funding sources did not play a role in the investigation. The Disclosure of Potential Conflicts of Interest forms are provided with the online version of the article ( http://links.lww.com/JBJS/I188 )., (Copyright © 2024 by The Journal of Bone and Joint Surgery, Incorporated.)

Mammography screening supported by deep learning-based artificial intelligence (AI) solutions can potentially reduce workload without compromising breast cancer detection accuracy, but the site of deployment in the workflow might be crucial. This retrospective study compared three simulated AI-integrated screening scenarios with standard double reading with arbitration in a sample of 249 402 mammograms from a representative screening population. A commercial AI system replaced the first reader (scenario 1: integrated AI first ), the second reader (scenario 2: integrated AI second ), or both readers for triaging of low- and high-risk cases (scenario 3: integrated AI triage ). AI threshold values were chosen based partly on previous validation and setting the screen-read volume reduction at approximately 50% across scenarios. Detection accuracy measures were calculated. Compared with standard double reading, integrated AI first showed no evidence of a difference in accuracy metrics except for a higher arbitration rate (+0.99%, P < .001). Integrated AI second had lower sensitivity (-1.58%, P < .001), negative predictive value (NPV) (-0.01%, P < .001), and recall rate (-0.06%, P = .04) but a higher positive predictive value (PPV) (+0.03%, P < .001) and arbitration rate (+1.22%, P < .001). Integrated AI triage achieved higher sensitivity (+1.33%, P < .001), PPV (+0.36%, P = .03), and NPV (+0.01%, P < .001) but lower arbitration rate (-0.88%, P < .001). Replacing one or both readers with AI seems feasible; however, the site of application in the workflow can have clinically relevant effects on accuracy and workload. Keywords: Mammography, Breast, Neoplasms-Primary, Screening, Epidemiology, Diagnosis, Convolutional Neural Network (CNN) Supplemental material is available for this article. Published under a CC BY 4.0 license.

Purpose: Patients with hypothalamic pathology often develop hypothalamic obesity, causing severe metabolic alterations resulting in increased morbidity and mortality. Treatments for hypothalamic obesity have not proven very effective, although the glucagon-like peptide-1 receptor agonist semaglutide has been shown to have positive effects. We examined semaglutide's effect on weight loss in a sample of patients with hypothalamic obesity., Methods: Four female patients with hypothalamic obesity resulting from treatment of craniopharyngiomas were treated with semaglutide for six months. Whole Body Dual-energy x-ray absorptiometry scans were performed, and blood samples drawn at baseline and after six months. Semaglutide dosages were increased monthly along with tracking of body weight and eating behavior (Three Factor Eating Questionnaire, TFEQ-R18)., Results: BMI was reduced in all cases, with an average of 7.9 BMI (range: 6.7 to 10.1) corresponding to a weight loss of 17.0% (range: 11.3-22.4%) or 20.2 kg (range 16.2 kg to 23.4 kg). We found a comparable reduction in total fat mass (17.2%, p = 0.006) and lean mass (16.0%, p = 0.05), whereas bone mass was unchanged (2.6%, p = 0.12). All cases reported an increase in energy levels, improved mobility and physical activity. Unfavorable eating behaviors were reduced after 1 month of treatment (emotional eating - 41 points, p = 0.02, uncontrolled eating - 23 points, p = 0.11). HbA1c and total cholesterol were significantly reduced (p = 0.014 for both)., Conclusion: Semaglutide is a promising and safe treatment option for HO, that improves eating behavior, reduces weight, and improves metabolic markers., (© 2024. The Author(s).)

The present study was conducted to assess the individual or combined effects of feeding dietary fat (whole-cracked rapeseed), nitrate, and 3-nitrooxypropanol (3-NOP) on protein profile, mineral composition, B vitamins, and nitrate residues in milk from dairy cows. A total of 48 Danish Holstein cows used in an 8 × 8 incomplete Latin square design were fed 8 factorially arranged diets: (30 or 63 g crude fat/kg DM) × (0 or 10 g nitrate/kg DM) × (0 or 80 mg 3-NOP/kg DM) over 6 periods of 21 d each. In each period, milk samples were collected from individual cows during the third week by pooling milk obtained from 4 consecutive milkings and analyzed for protein profile, including protein modifications, mineral composition, riboflavin, cobalamin, and presence of nitrate residues. Fat supplementation led to an increase in the phosphorylation degree of α S1 -CN by 8.5% due to a decreased relative proportion of α S1 -CN 8P and an increased relative proportion of α S1 -CN 9P and further to a decrease in the relative proportion of α S2 -CN by 2.4%. Additionally, fat supplementation decreased the relative proportions of glycosylated and unglycosylated forms of κ-CN, consequently leading to a 3.6% decrease in total κ-CN. In skim milk, K, Ca, P, and Mg concentrations were altered by individual use of fat, nitrate, and 3-NOP. Feeding nitrate resulted in a 5.4% increase in riboflavin concentration in milk, whereas supplementing 3-NOP increased the cobalamin concentration in milk by 21.1%. The nitrate concentration in milk was increased upon feeding nitrate, but this increased concentration was well below the maximum permissible limit of nitrate in milk (<50 mg/L). Overall, no major changes were observed in milk protein, and mineral compositions by feeding fat, nitrate, and 3-NOP to dairy cows, but the increased riboflavin and cobalamin concentrations by nitrate and 3-NOP, respectively, could be of beneficial nutritional value for milk consumers., (© 2024, The Authors. Published by Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

This study explores the disulfide bridges present in the human milk proteome by a novel approach permitting both positional identification and relative quantification of the disulfide bridges. Human milk from six donors was subjected to trypsin digestion without reduction. The digested human milk proteins were analyzed by nanoLC-timsTOF Pro combined with data analysis using xiSEARCH. A total of 85 unique disulfide bridges were identified in 25 different human milk proteins. The total relative abundance of disulfide bridge-containing peptides constituted approximately 5% of the total amount of tryptic-peptides. Seven inter-molecular disulfide bridges were identified between either α-lactalbumin and lactotransferrin (5) or α S1 -casein and κ-casein (2). All cysteines involved in the observed disulfide bridges of α-lactalbumin and lactotransferrin were mapped onto protein models using AlphaFold2 Multimer to estimate the length of the observed disulfide bridges. The lengths of the disulfide bridges of lactotransferrin indicate a potential for multi- or poly-merization of lactotransferrin. The high number of intramolecular lactotransferrin disulfide bridges identified, suggests that these are more heterogeneous than previously presumed. SIGNIFICANCE: Disulfide-bridges in the human milk proteome are an often overseen post-transaltional modification. Thus, mapping the disulfide-bridges, their positions and relative abundance, are valuable new knowledge needed for an improved understanding of human milk protein behaviour. Although glycosylation and phosphorylation have been described, even less information is available on the disulfide bridges and the disulfide-bridge derived protein complexes. This is important for future work in precision fermentation for recombinant production of human milk proteins, as this will highlight which disulfide-bridges are naturally occouring in human milk proteins. Further, this knowledge would be of value for the infant formula industry as it provides more information on how to humanize bovine-milk based infant formula. The novel method developed here can be broadly applied in other biological systems as the disulfid-brigdes are important for the structure and functionality of proteins., Competing Interests: Declaration of competing interest S.D-H.N. currently holds a position at Arla Foods Ingredients Group P/S Viby J. The remaining authors declare no conflict of interest. The funders had no role in the design of the study, in the collection, analysis or interpretation of data nor in writing the manuscript or in the decision to publish the results., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Objectives: To validate an AI system for standalone breast cancer detection on an entire screening population in comparison to first-reading breast radiologists., Materials and Methods: All mammography screenings performed between August 4, 2014, and August 15, 2018, in the Region of Southern Denmark with follow-up within 24 months were eligible. Screenings were assessed as normal or abnormal by breast radiologists through double reading with arbitration. For an AI decision of normal or abnormal, two AI-score cut-off points were applied by matching at mean sensitivity (AI sens ) and specificity (AI spec ) of first readers. Accuracy measures were sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and recall rate (RR)., Results: The sample included 249,402 screenings (149,495 women) and 2033 breast cancers (72.6% screen-detected cancers, 27.4% interval cancers). AI sens had lower specificity (97.5% vs 97.7%; p < 0.0001) and PPV (17.5% vs 18.7%; p = 0.01) and a higher RR (3.0% vs 2.8%; p < 0.0001) than first readers. AI spec was comparable to first readers in terms of all accuracy measures. Both AI sens and AI spec detected significantly fewer screen-detected cancers (1166 (AI sens ), 1156 (AI spec ) vs 1252; p < 0.0001) but found more interval cancers compared to first readers (126 (AI sens ), 117 (AI spec ) vs 39; p < 0.0001) with varying types of cancers detected across multiple subgroups., Conclusion: Standalone AI can detect breast cancer at an accuracy level equivalent to the standard of first readers when the AI threshold point was matched at first reader specificity. However, AI and first readers detected a different composition of cancers., Clinical Relevance Statement: Replacing first readers with AI with an appropriate cut-off score could be feasible. AI-detected cancers not detected by radiologists suggest a potential increase in the number of cancers detected if AI is implemented to support double reading within screening, although the clinicopathological characteristics of detected cancers would not change significantly., Key Points: • Standalone AI cancer detection was compared to first readers in a double-read mammography screening population. • Standalone AI matched at first reader specificity showed no statistically significant difference in overall accuracy but detected different cancers. • With an appropriate threshold, AI-integrated screening can increase the number of detected cancers with similar clinicopathological characteristics., (© 2023. The Author(s).)

As glycosylations are difficult to analyze, their roles and effects are poorly understood. Glycosylations in human milk (HM) differ across lactation. Glycosylations can be involved in antimicrobial activities and may serve as food for beneficial microorganisms. This study aimed to identify and analyze O-linked glycans in HM by high-throughput mass spectrometry. 184 longitudinal HM samples from 66 donors from day 3 and months 1, 2, and 3 postpartum were subjected to a post-translational modification specific enrichment-based strategy using TiO 2 and ZrO 2 beads for O-linked glycopeptide enrichment. β-CN was found to be a major O-linked glycoprotein, additionally, α S1 -CN, κ-CN, lactotransferrin, and albumin also contained O-linked glycans. As glycosyltransferases and glycosidases are involved in assembling the glycans including O-linked glycosylations, these were further investigated. Some glycosyltransferases and glycosidases were found to be significantly decreasing through lactation, including two O-linked glycan initiator enzymes (GLNT1 and GLNT2). Despite their decrease, the overall level of O-linked glycans remained stable in HM over lactation. Three different motifs for O-linked glycosylation were enriched in HM proteins: Gly-Xxx-Xxx-Gly-Ser/Thr, Arg-Ser/Thr and Lys-Ser/Thr. Further O-linked glycan motifs on β-CN were observed to differ between intact proteins and endogenous peptides in HM., Competing Interests: Declaration of competing interest S.D-H.N. and K.O.P. currently hold positions in Arla Foods Ingredients. The remaining authors declare no conflicts of interest. The funders had no role in the design of the study; the collection, analysis, or interpretation of data; the writing of the manuscript; or the decision to publish the results., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

In line with the peer reviewers comments, the authors have added highlights in stead of an abstract. It was felt that it was better able to capture the findings and is more in line with the paper's target audience., 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., (© 2024 The Authors. Published by Elsevier Ltd.)

Milk proteins produced by lactating cells isolated from bovine mammary tissue can offer a sustainable solution to the high protein demand of a global growing population. Serum is commonly added to culture systems to provide compounds necessary for optimal growth and function of the cells. However, in a cellular agricultural context, its usage is desired to be decreased. This study aims at examining the minimum level of fetal bovine serum (FBS) required for the growth and functionality of bovine mammary epithelial cells (MECs). The cells were isolated from dairy cows in early and mid-lactation and cultured in reduced concentrations of FBS (10%, 5%, 1.25%, and 0%). Real-time cell analysis showed a significant effect of lactation stage on growth rate and 5% FBS resulted in similar growth rate as 10% while 0% resulted in the lowest. The effect of reducing FBS on cell functionality was examined by studying the expressions of selected marker genes involved in milk protein and fat synthesis, following differentiation. The gene expressions were not affected by the level of FBS. A reduction of FBS in the culture system of MEC, at least down to 5%, does not assert any negative effect on the growth and expression levels of studied genes. As the first attempt in developing an in-vitro model for milk component production using MEC, our results demonstrate the potential of MEC to endure FBS-reduced conditions., (© 2024 International Federation of Cell Biology.)

In this commentary, we advocate for the wider implementation of integrated care models for NCDs within humanitarian preparedness, response, and resilience efforts. Since experience and evidence on integrated NCD care in humanitarian settings is limited, we discuss potential benefits, key lessons learned from other settings, and lessons from the integration of other conditions that may be useful for stakeholders considering an integrated model of NCD care. We also introduce our ongoing project in North Lebanon as a case example currently undergoing parallel tracks of program implementation and process evaluation that aims to strengthen the evidence base on implementing an integrated NCD care model in a crisis setting., 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., (© 2024 The Authors. Published by Elsevier Ltd.)

The market for plant-based drinks (PBDs) is experiencing a surge in consumer demand, especially in Western societies. PBDs are a highly processed food product, and little is known about this relatively new food product category when compared to bovine milk. In the present study, the storage stability, proteolysis and generation of free amino acids were investigated in commercially available PBDs over the course of a one-year storage period. Generally, pH, color and protein solubility were found to be stable in the PBDs during storage, except for the pea-based product, which showed less protein solubility after storage. The pea-based drinks also had higher initial levels of free N-terminals prior to storage compared with levels for the other plant-based drinks, as well as significantly increasing levels of total free, and especially bitter free, amino acids. The development of free amino acids in the oat-based drink indicated that the released amino acids could be involved in various reactions such as the Maillard reaction during the storage period.

Background: Artificial intelligence (AI) systems are proposed as a replacement of the first reader in double reading within mammography screening. We aimed to assess cancer detection accuracy of an AI system in a Danish screening population., Methods: We retrieved a consecutive screening cohort from the Region of Southern Denmark including all participating women between Aug 4, 2014, and August 15, 2018. Screening mammograms were processed by a commercial AI system and detection accuracy was evaluated in two scenarios, Standalone AI and AI-integrated screening replacing first reader, with first reader and double reading with arbitration (combined reading) as comparators, respectively. Two AI-score cut-off points were applied by matching at mean first reader sensitivity (AI sens ) and specificity (AI spec ). Reference standard was histopathology-proven breast cancer or cancer-free follow-up within 24 months. Coprimary endpoints were sensitivity and specificity, and secondary endpoints were positive predictive value (PPV), negative predictive value (NPV), recall rate, and arbitration rate. Accuracy estimates were calculated using McNemar's test or exact binomial test., Results: Out of 272,008 screening mammograms from 158,732 women, 257,671 (94.7%) with adequate image data were included in the final analyses. Sensitivity and specificity were 63.7% (95% CI 61.6%-65.8%) and 97.8% (97.7-97.8%) for first reader, and 73.9% (72.0-75.8%) and 97.9% (97.9-98.0%) for combined reading, respectively. Standalone AI sens showed a lower specificity (-1.3%) and PPV (-6.1%), and a higher recall rate (+ 1.3%) compared to first reader (p < 0.0001 for all), while Standalone AI spec had a lower sensitivity (-5.1%; p < 0.0001), PPV (-1.3%; p = 0.01) and NPV (-0.04%; p = 0.0002). Compared to combined reading, Integrated AI sens achieved higher sensitivity (+ 2.3%; p = 0.0004), but lower specificity (-0.6%) and PPV (-3.9%) as well as higher recall rate (+ 0.6%) and arbitration rate (+ 2.2%; p < 0.0001 for all). Integrated AI spec showed no significant difference in any outcome measures apart from a slightly higher arbitration rate (p < 0.0001). Subgroup analyses showed higher detection of interval cancers by Standalone AI and Integrated AI at both thresholds (p < 0.0001 for all) with a varying composition of detected cancers across multiple subgroups of tumour characteristics., Conclusions: Replacing first reader in double reading with an AI could be feasible but choosing an appropriate AI threshold is crucial to maintaining cancer detection accuracy and workload., (© 2023. The Author(s).)

Background: Preventive health checks are assumed to reduce the risk of the development of cardio-metabolic disease in the long term. Although no solid evidence of effect is shown on health checks targeting the general population, studies suggest positive effects if health checks target people or groups identified at risk of disease. The aim of this study is to explore why and how targeted preventive health checks work, for whom they work, and under which circumstances they can be expected to work., Methods: The study is designed as a realist synthesis that consists of four phases, each including collection and analysis of empirical data: 1) Literature search of systematic reviews and meta-analysis, 2) Interviews with key-stakeholders, 3) Literature search of qualitative studies and grey literature, and 4) Workshops with key stakeholders and end-users. Through the iterative analysis we identified the interrelationship between contexts, mechanisms, and outcomes to develop a program theory encompassing hypotheses about targeted preventive health checks., Results: Based on an iterative analysis of the data material, we developed a final program theory consisting of seven themes; Target group; Recruitment and participation; The encounter between professional and participants; Follow-up activities; Implementation and operation; Shared understanding of the intervention; and Unintended side effects. Overall, the data material showed that targeted preventive health checks need to be accessible, recognizable, and relevant for the participants' everyday lives as well as meaningful to the professionals involved. The results showed that identifying a target group, that both benefit from attending and have the resources to participate pose a challenge for targeted preventive health check interventions. This challenge illustrates the importance of designing the recruitment and intervention activities according to the target groups particular life situation., Conclusion: The results indicate that a one-size-fits-all model of targeted preventive health checks should be abandoned, and that intervention activities and implementation depend on for whom and under which circumstances the intervention is initiated. Based on the results we suggest that future initiatives conduct thorough needs assessment as the basis for decisions about where and how the preventive health checks are implemented., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Children with attention-deficit/hyperactivity disorder (ADHD) can be more stress-vulnerable, and thereby, it has been suggested, prone to develop functional somatic symptoms (FSS) compared to their peers. In this paper, using data from 160 children aged 3-7 years with ADHD from the D'SNAPP study, a randomized controlled trial testing a parent training intervention, we addressed a number of questions about the role of FSS in ADHD. First, are FSS levels higher in an ADHD sample than in the children of the general population. Second, do FSS levels predict psychopathology and health-related quality of life (HRQoL) in ADHD samples. Third, does FSS levels moderate the effect of parent training on ADHD symptoms. We found that preschoolers with ADHD experienced more severe FSS than a general population-based sample (18.80% vs. 2.11%). Severe FSS were associated with increased psychopathology and impaired daily function and lower HRQoL. Level of baseline FSS did not moderate the effect of parent training on ADHD. FSS in preschool children with ADHD is associated with impaired daily functioning, but further research is warranted to determine the clinical impact of FSS in children with ADHD., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany.)

The presence of proteases and their resulting level of activity on human milk (HM) proteins may aid in the generation of indigenous peptides as part of a pre-digestion process, of which some have potential bioactivity for the infant. The present study investigated the relative abundance of indigenous peptides and their cleavage products in relation to the abundance of observed proteases and protease inhibitors. The proteomes and peptidomes in twelve HM samples, representing six donors at lactation months 1 and 3, were profiled. In the proteome, 39 proteases and 29 protease inhibitors were identified in 2/3 of the samples. Cathepsin D was found to be present in higher abundance in the proteome compared with plasmin, while peptides originating from plasmin cleavage were more abundant than peptides from cathepsin D cleavage. As both proteases are present as a system of pro- and active- forms, their activation indexes were calculated. Plasmin was more active in lactation month 3 than month 1, which correlated with the total relative abundance of the cleavage product ascribed to plasmin. By searching the identified indigenous peptides in the milk bioactive peptide database, 283 peptides were ascribed to 10 groups of bioactivities. Antimicrobial peptides were significantly more abundant in month 1 than month 3; this group comprised 103 peptides, originating from the β-CN C-terminal region.

The consumption of plant-based drinks is increasing, but they represent a product category normally with lower protein content as compared with bovine milk. Furthermore, the products are highly processed and, therefore, the proteins in this product category may carry a significant processing history. In the present study, a series of 17 freshly produced, commercially available plant-based drinks were benchmarked according to protein-quality parameters. The plant-based drinks represented different plant sources, as well as some mixed products, and were investigated relative to composition, aggregate sizes, presence of non-reducible proteins complexes, and level of processing-induced markers in the proteins. Processing-induced changes in the proteins were determined by a newly developed cocktail method, determining markers related to Maillard and dehydroalanine pathways, as well as intact lysine by triple quadrupole-multiple reaction monitoring-mass spectrometry. It was found that all drinks contained non-reducible protein complexes, but specifically, oat-based drinks represented the largest span contents of processing-induced markers within the proteins, which may relate to their inherent processing histories. Furthermore, it was shown that in products containing added sugar, Maillard reaction-related processing markers were increased over the dehydroalanine pathway.

AprX is an alkaline metalloprotease produced by Pseudomonas spp. and encoded by its initial gene of the aprX-lipA operon. The intrinsic diversity among Pseudomonas spp. regarding their proteolytic activity is the main challenge for the development of accurate methods for spoilage prediction of ultra-high temperature (UHT) treated milk in the dairy industry. In the present study, 56 Pseudomonas strains were characterized by assessing their proteolytic activity in milk before and after lab-scale UHT treatment. From these, 24 strains were selected based on their proteolytic activity for whole genome sequencing (WGS) to identify common genotypic characteristics that correlated with the observed variations in proteolytic activity. Four groups (A1, A2, B and N) were determined based on operon aprX-lipA sequence similarities. These alignment groups were observed to significantly influence the proteolytic activity of the strains, with an average proteolytic activity of A1 > A2 > B > N. The lab-scale UHT treatment did not significantly influence their proteolytic activity, indicating a high thermal stability of proteases among strains. Amino acid sequence variation of biologically-relevant motifs in the AprX sequence, namely the Zn 2+ -binding motif at the catalytic domain and the C-terminal type I secretion signaling mechanism, were found to be highly conserved within alignment groups. These motifs could serve as future potential genetic biomarkers for determination of alignment groups and thereby strain spoilage potential., Competing Interests: Declaration of competing interest Piccini, V., Rauh, V. and Xiao, Y. are employed by Arla Foods Amba. The other authors declare no conflict of interest., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Aim: To evaluate the cost-effectiveness of surgical treatment with reverse total shoulder arthroplasty (RTSA) compared with open reduction and internal fixation (ORIF) with a locking plate for patients 65-85 years old with a displaced proximal humerus fracture., Methods: A cost-utility analysis was conducted alongside a multicenter randomized controlled trial, taking a health care perspective. A total of 124 patients with displaced proximal humerus fractures were randomized to treatment with RTSA (n = 64) or ORIF (n = 60) during a 2-year period. The outcome measure was quality-adjusted life years derived from the generic questionnaire 15D in an intention to treat population. The results were expressed as incremental cost-effectiveness ratios, and a probabilistic sensitivity analysis was performed to account for uncertainty in the analysis., Results: At 2 years, 104 patients were eligible for analyses. The mean quality-adjusted life year was 1.24 (95% confidence interval: 1.21-1.28) in the RTSA group and 1.26 (95% confidence interval: 1.22-1.30) in the ORIF group. The mean cost in the RTSA group (€36.755 [€17,654-€55,855]) was higher than that in the ORIF group (€31.953 [€16,226-€47,279]). Using incremental cost-effectiveness ratio, ORIF was the dominant treatment. When using a probabilistic sensitivity analysis with 1000 replications, the plots were centered around origo. This indicates that there is no significant difference in cost or effect., Conclusion: In the cost-utility analysis of treatment of displaced proximal humeral fractures, there were no differences between RTSA and ORIF., (Copyright © 2022 Journal of Shoulder and Elbow Surgery Board of Trustees. Published by Elsevier Inc. All rights reserved.)

The objective of the study was to quantify the effects on dry matter intake (DMI), nutrient digestibility, gas exchange, milk production, and milk quality in dairy cows fed fresh grass harvested at different maturity stages. Sixteen Danish Holstein cows in mid-lactation were divided into 4 blocks and used in 4 incomplete 4 × 2 Latin squares with 2 periods of 21 d. The cows received 1 of 4 treatments in each period, resulting in 8 cows per treatment, as follows: grass-clover silage supplemented with 6 kg/d concentrate pellets (SILc), fresh grass harvested at late maturity stage supplemented with 6 kg/d concentrate pellets (LATc), fresh grass harvested at late maturity stage (LAT), and fresh grass harvested at early maturity stage (ERL). The cows were housed in tiestalls and milked twice daily. The cows had ad libitum access to the forage, and concentrate pellets were divided into equal amounts and fed separately in the morning and afternoon. Fecal samples were collected to determine apparent total-tract digestibility, and samples of rumen fluid were collected for determination of short chain fatty acid composition. Halters were used for measuring eating and rumination time. Gas exchange was measured in open-circuit respiration chambers. Total DMI was higher in LATc and ERL (16.9 ± 0.45 and 15.5 ± 0.39 kg/d, respectively) compared with LAT (14.1 ± 0.42 kg/d). Relative to SILc, cows fed fresh grass experienced a convex pattern in DMI during the experiment. The changes in DMI were related to changes in leaf to stem ratio, fiber concentration, and organic matter digestibility determined in vitro in samples of the fresh grass harvested throughout the experiment. The apparent total-tract digestibility of organic matter was higher in SILc and LAT compared with LATc. Methane yield was lower for LATc compared with LAT (19.5 ± 0.61 vs. 22.6 ± 0.55 g of CH 4 /kg of DMI), and was not different between LAT and ERL. Compared with LAT, milk yield was higher for ERL (21.1 ± 1.14 vs. 23.4 ± 1.11 kg/d) and energy-corrected milk (ECM) yield was higher for LATc (21.5 ± 0.99 vs. 25.3 ± 1.03 kg/d). We detected no differences in milk or ECM yield between SILc and LATc. Milk protein yield was higher and milk fat concentration was lower in LATc compared with LAT. The fatty acid percentages of ∑C4-C14:1 and ∑C16 in milk were higher for SILc compared with LATc, signifying pronounced de novo synthesis. The n-6:n-3 ratio in milk fatty acids was lower for SILc and LAT compared with LATc, indicating improved nutritional quality for SILc and LAT. However, retinol concentration in milk was lower in SILc compared with all other treatments. The study implies that feeding silage instead of fresh grass has no effect on DMI, ECM yield, or CH 4 yield, and that concentrate supplementation can increase milk production, affects milk quality, and reduces the effect on climate, whereas feeding less mature grass increases DMI and milk yield, but has no effect on CH 4 yield., (The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Interactive biorobotics provides unique experimental potential to study the mechanisms underlying social communication but is limited by our ability to build expressive robots that exhibit the complex behaviours of birds and small mammals. An alternative to physical robots is to use virtual environments. Here, we designed and built a modular, audio-visual 2D virtual environment that allows multi-modal, multi-agent interaction to study mechanisms underlying social communication. The strength of the system is an implementation based on event processing that allows for complex computation. We tested this system in songbirds, which provide an exceptionally powerful and tractable model system to study social communication. We show that pair-bonded zebra finches (Taeniopygia guttata) communicating through the virtual environment exhibit normal call timing behaviour, males sing female directed song and both males and females display high-intensity courtship behaviours to their mates. These results suggest that the environment provided is sufficiently natural to elicit these behavioral responses. Furthermore, as an example of complex behavioral annotation, we developed a fully unsupervised song motif detector and used it to manipulate the virtual social environment of male zebra finches based on the number of motifs sung. Our virtual environment represents a first step in real-time automatic behaviour annotation and animal-computer interaction using higher level behaviours such as song. Our unsupervised acoustic analysis eliminates the need for annotated training data thus reducing labour investment and experimenter bias., (© 2022. The Author(s).)

Ultra-high temperature (UHT) processing of milk can result in protein changes during storage; however, the progress of dehydroalanine (DHA) mediated protein cross-linking and Maillard reactions in relation to the sediment formation have not been investigated previously. Liquid chromatography-mass spectrometry, based on multiple reaction monitoring (MRM), was used to absolutely quantify concentrations of furosine, N-ε-(carboxyethyl)lysine (CEL), N-ε-(carboxymethyl)lysine (CML), lanthionine (LAN) and lysinoalanine (LAL) in skim milk and sediment of UHT milk produced from raw milk with either small or large casein micelles. The results showed a higher molar proportion of the advanced stage Maillard reaction products CEL and CML in the sediment, compared to early stage Maillard reaction product furosine, whereas furosine was predominant in the skim milk. Both LAL and LAN increased during storage in the skim milk phase, however only LAL was identified in the sediment. The milk pool with large native casein micelles, known to have a higher percentage of sedimentation, contained higher proportions of furosine, CEL, CML and LAL in the sediment compared to milk with smaller native casein micelles. The study demonstrates the potential contribution of processing-induced protein-protein interactions to sedimentation in UHT milk during storage.

Background: Having an unhealthy lifestyle is associated with a higher risk of developing lifestyle-related diseases. Current evidence suggests that interventions targeting health-risk behaviors can help people improve their lifestyles and prevent lifestyle-related diseases. However, preventive programs are often challenged by low participation rates. Reasons for non-participation include lack of time and/or interest, and/or no perceived need for lifestyle intervention. This study explores causes for non-participation in a sample of people who chose not to take up a targeted preventive program (TOF pilot2 study). Patient-reported reasons as well as sociodemographic characteristics and lifestyle factors are in focus., Methods: A total of 4633 patients from four Danish GP clinics received an invitation to take part in the TOF pilot2 study. Patients who chose not to participate in the TOF pilot2 study were asked to fill in a questionnaire concerning reasons for non-participation, lifestyle, BMI and self-rated health. Descriptive analyses were used to summarize the results., Results: A total of 2462 patients (53.1%) chose not to participate in the TOF pilot2 study. Among these, 84 (3.4%) answered the full questionnaire on reasons for not participating, lifestyle, BMI and self-rated health. The most common reasons for non-participation were lack of time, having an already healthy lifestyle, and feeling healthy. Based on their self-reported lifestyle 45 (53.6%) of the non-participants had one or more health-risk behaviors including smoking, unhealthy diet, BMI ≥ 35 and/or sedentary lifestyle and were therefore eligible to receive the targeted intervention at the GP or the MHC in the original TOF pilot2 study., Conclusion: When planning future preventive programs it is important to know the main reasons for patients to not participate. This study provides rare insight into why people opt out of health interventions and advances the evidence base in this area. Our results may inform efforts to better involve these patients in preventive health programs., Trial Registration: Trial registration number: NCT02797392 ., (© 2022. The Author(s).)

The walking system of the stick insect is one of the most thoroughly described invertebrate systems. We know a lot about the role of sensory input in the control of stepping of a single leg. However, the neuronal organization and connectivity of the central neural networks underlying the rhythmic activation and coordination of leg muscles still remain elusive. It is assumed that these networks can couple in the absence of phasic sensory input due to the observation of spontaneous recurrent patterns (SRPs) of coordinated motor activity equivalent to fictive stepping-phase transitions. Here we sought to quantify the phase of motor activity within SRPs in the isolated and interconnected meso- and meta-thoracic ganglia. We show that SRPs occur not only in the meso-, but also in the metathoracic ganglia of the stick insect, discovering a qualitative difference between them. We construct a network based on neurophysiological data capable of reproducing the measured SRP phases to investigate this difference. By comparing network output to the biological measurements we confirm the plausibility of the architecture and provide a hypothesis to account for these qualitative differences. The neural architecture we present couples individual central pattern generators to reproduce the fictive stepping-phase transitions observed in deafferented stick insect preparations after pharmacological activation, providing insights into the neural architecture underlying coordinated locomotion., 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 Strohmer, Mantziaris, Kynigopoulos, Manoonpong, Larsen and Büschges.)

Milk with different κ-casein (CN) phenotypes has previously been found to influence its gastric digestion rate. Therefore, the aim of the present study is to disentangle contributions of genetic variation and its related sialylation on the in vitro digestion process of κ-CN. Accordingly, κ-CN was purified from milk representing homozygous cows with κ-CN phenotypes AA, BB, or EE and used as substrate molecules in model studies using the INFOGEST 2.0 in vitro static digestion model. Furthermore, the effect of removal of the terminal sialic acids present on the O-linked oligosaccharides of the purified κ-CN A, B, and E protein variants were studied by desialylation enzymatic assays. The κ-CN proteins were purified by reducing anion exchange chromatography with purities of variants A, B, and E of 93.0, 97.1, and 90.0%, respectively. Protein degradations of native and desialylated κ-CN isolates in gastric and intestinal phases were investigated by sodium dodecyl sulfate-PAGE, degree of hydrolysis (DH), and liquid chromatography electrospray ionization mass spectrometry. It was shown that after purification, the κ-CN molecules reassembled into multimer states, which then constituted the basis for the digestion studies. As assessed by DH, purified variants A and E were found to exhibit faster in vitro digestion rates in both gastric and intestinal phases compared with variant B. Desialylation increased both gastric and intestinal digestion rates for all variants, as measured by DH. In the gastric phase, desialylation promoted digestion of variant B at a rate comparable with native variants A and E, whereas in the intestinal phase, desialylation of variant B promoted better digestion than native A or E. Taken together, the results confirm that low glycosylation degree of purified κ-CN promotes faster in vitro digestion rates, and that desialylation of the O-linked oligosaccharides further promotes digestion. This finding could be applied to produce dairy products with enhanced digestibility., (The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

A liquid chromatography-mass spectrometry method based on multiple reaction monitoring (MRM) was developed for the simultaneous quantification of markers representing two potentially competing pathways, the Maillard reaction and the dehydroalanine pathway. The two pathways involve the same residues in the proteins to some extent, namely, the essential amino acid lysine, as well as free-amino terminals available on proteins and polypeptides, competition between the two pathways in food systems may occur. The developed method comprises the following markers of the Maillard reaction: furosine, N-ε-(carboxyethyl)lysine (CEL) and N-ε-(carboxymethyl)lysine (CML), together with the dehydroalanine reaction pathway markers; lanthionine (LAN) and lysinoalanine (LAL), as well as lysine itself. The validated method was then used for the absolute quantification of heat-induced protein modifications in model systems of micellar casein and whey protein isolates (MCI and WPI, respectively) in the presence or absence of lactose. As expected, the Maillard reaction markers furosine, CEL and CML increased during the applied heat treatment in the presence of lactose, whereas the dehydroalanine markers, LAN and LAL increased with heating in both MCI and WPI, both in the presence and absence of lactose, although at lower levels in the presence of lactose, confirming the competing state of the two pathways.

Variations in the phosphorylation and glycosylation patterns of the common κ-casein (CN) variants A and B have been explored, whereas studies on variant E heterogeneity are scarce. This study reports for the first time the detailed phosphorylation and glycosylation pattern of the κ-CN variant E in comparison with variants A and B. Individual cow milk samples representing κ-CN genotype EE (n = 12) were obtained from Swedish Red cows, and the natural posttranslational modifications of its κ-CN were identified and quantified by liquid chromatography-electrospray mass spectrometry. In total, 12 unique isoform masses of κ-CN variant E were identified. In comparison, AA and BB milk consisted of 14 and 17 unique isoform masses, respectively. The most abundant κ-CN E isoform detected in the EE milk was the monophosphorylated, unglycosylated [1P 0G, ∼70%; where P indicates phosphorylation from single to triple phosphorylation (1-3P), and G indicates glycosylation from single to triple glycosylation (1-3G)] form, followed by diphosphorylated, unglycosylated (2P 0G, ∼12%) form, resembling known patterns from variants A and B. However, a clear distinction was the presence of the rare triphosphorylated, nonglycosylated (3P 0G, ∼0.05%) κ-CN isoform in the EE milk. All isoforms detected in variant E were phosphorylated, giving a phosphorylation degree of 100%. This is comparable with the phosphorylation degree of variants A and B, being also almost 100%, though with very small amounts of nonphosphorylated, glycosylated isoforms detected. The glycosylation degree of variant E was found to be around 17%, a bit higher than observed for variant B (around 14%), and higher than variant A (around 7%). Among glycosylation, the glycan e was the most common type identified for all 3 variants, followed by c/d (straight and branched chain trisaccharides, respectively), and b. In contrast to κ-CN variants A and B, no glycan of type a was found in variant E. Taken together, this study shows that the posttranslational modification pattern of variant E resembles that of known variants to a large extent, but with subtle differences., (The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Several factors influence the composition of milk. Among these, genetic variation within and between cattle breeds influences milk protein composition, protein heterogeneity, and their posttranslational modifications. Such variations may further influence technological properties, which are of importance for the utilization of milk into dairy products. Furthermore, these potential variations may also facilitate the production of differentiated products (e.g., related to specific breeds or specific genetic variants). The objective of this study was to investigate the genetic variation and relative protein composition of the major proteins in milk from 6 native Norwegian dairy breeds representing heterogeneity in geographical origin, using the modern Norwegian breed, Norwegian Red, as reference. In total, milk samples from 144 individual cows were collected and subjected to liquid chromatography-electrospray ionization/mass spectrometry-based proteomics for identification of genetic and posttranslational modification isoforms of the 4 caseins (α S1 -CN, α S2 -CN, β-CN, κ-CN) and the 2 most abundant whey proteins (α-lactalbumin and β-lactoglobulin). Relative quantification of these proteins and their major isoforms, including phosphorylations of α S1 -CN and glycosylation of κ-CN, were determined based on UV absorbance. The presence and frequency of genetic variants of the breeds were found to be very diverse and it was possible to identify rare variants of the CN, which, to our knowledge, have not been identified in these breeds before. Thus, α S1 -CN variant D was identified in low frequency in 3 of the 6 native Norwegian breeds. In general, α S1 -CN was found to be quite diverse between the native breeds, and the even less frequent A and C variants were furthermore detected in 1 and 5 of the native breeds, respectively. The α S1 -CN variant C was also identified in samples from the Norwegian Red cattle. The variant E of κ-CN was identified in 2 of the native Norwegian breeds. Another interesting finding was the identification of α S2 -CN variant D, which was found in relatively high frequencies in the native breeds. Diversity in more common protein genetic variants were furthermore observed in the protein profiles of the native breeds compared with milk from the high-yielding Norwegian Reds, probably reflecting the more diverse genetic background between the native breeds., (The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Casein (CN) micelles will coagulate in the stomach after ingestion, which is similar to the cheesemaking process. Although genetic variants of bovine proteins, especially κ-CN, have been confirmed to influence the coagulation properties of the CN micelle, its influence on milk digestibility has not been revealed yet. This study aimed to investigate how genetic variants, glycosylation degree of κ-CN, and CN micelle size influence digestion rates during in vitro gastrointestinal digestion. Three milk pools, representing κ-CN phenotypes of either AA, BB, or AB composition were prepared from milk of individual Danish Holstein cows representing these different genotypes. In vitro digestion of the 3 milk pools, AA, BB, or AB, was investigated by sodium dodecyl sulfate-PAGE, liquid chromatography-mass spectrometry, and degree of hydrolysis. The results showed that κ-CN AA milk had faster digestion rate in the gastric phase compared with BB and AB milks, whereas only small differences were apparent in the intestinal digestion phase. The results further documented that the milk pools representing κ-CN phenotypes BB and AB had comparable overall glycosylation degrees (50.9% and 50.0%, respectively) and higher than that of the AA milk pool (46.9%). Further, the AA milk pool was associated with larger CN micelles. These differences in CN micelle sizes and glycosylation degrees can be part of underlying explanations for the differential in vitro digestion rates observed between the AA, BB, and AB κ-CN milk pools., (Copyright © 2021 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Little is known about the extent of variation and activity of naturally occurring milk glycosidases and their potential to degrade milk glycans. A multi-omics approach was used to investigate the relationship between glycosidases and important bioactive compounds such as free oligosaccharides and O -linked glycans in bovine milk. Using 4-methylumbelliferone (4-MU) assays activities of eight indigenous glycosidases were determined, and by mass spectrometry and 1 H NMR spectroscopy various substrates and metabolite products were quantified in a subset of milk samples from eight native North European cattle breeds. The results showed a clear variation in glycosidase activities among the native breeds. Interestingly, negative correlations between some glycosidases including β-galactosidase, N -acetyl-β- d -glucosaminidase, certain oligosaccharide isomers as well as O -linked glycans of κ-casein were revealed. Further, a positive correlation was found for free fucose content and α-fucosidase activity (r = 0.37, p -value < 0.001) indicating cleavage of fucosylated glycans in milk at room temperature. The results obtained suggest that milk glycosidases might partially degrade valuable glycans, which would result in lower recovery of glycans and thus represent a loss for the dairy ingredients industry if these activities are pronounced.

Variations in major milk minerals, proteins, and their posttranslational modifications are largely under genetic influence, whereas the effect of nongenetic factors is less studied. Through a controlled feeding experiment (incomplete balanced Latin square design), the effect of concentrate mixtures, based on fava beans, rapeseed meal, or soybean meal as main P and protein sources, on milk composition was examined under typical Danish management conditions. Concentrations of P, Ca, and Mg, together with proteomics for relative quantification of major milk proteins and their isoforms, were analyzed in milk samples from 24 cows sampled in 4 periods. Each cow was fed 1 of the 3 diets in each period with or without addition of exogenous phytase. Cows were blocked by lactation stage into early and mid-lactation (23.3 ± 6.7 and 176 ± 15 d in milk, respectively, at the beginning of the experiment, mean ± standard deviation). Significant effects of feed concentrate mixture were observed for milk protein concentration, milk urea nitrogen, citrate, and the percentage of mixed and preformed fatty acids as well as mineral composition, and their distributions within micellar or serum phases. Furthermore, relative contents of α S1 -casein (CN) 9P form and unglycosylated κ-CN and thereby phosphorylation degree of α S1 -CN (PD) and the glycosylation degree of κ-CN were found to be significantly affected by these diets. To our knowledge, we are the first to document that feed concentrate mixture can affect the relative concentrations of α S1 -CN phosphorylation isoforms in milk, and the results suggested an effect on α S1 -CN 9P and PD, but not on α S1 -CN 8P. Furthermore, although only significant for α S1 -CN 8P, we found a lower relative concentration of α S1 -CN 8P and higher α S1 -CN 9P (and thus higher PD) in milk from cows in mid compared with early lactation. Also, protein concentration and concentration of Mg in skim milk and serum as well as relative concentration of α-lactalbumin were found to be significantly affected by lactation stage. Addition of dietary exogenous phytase only had a minor effect on milk composition or functionality with significant effect detected for α-lactalbumin and micellar Mg concentration., (Copyright © 2021 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Aims: The aim of this study was to derive and validate a risk prediction model with nationwide coverage to predict the individual and population-level risk of cardiovascular disease (CVD)., Methods and Results: All 2.98 million Danish residents aged 30-85 years free of CVD were included on 1 January 2014 and followed through 31 December 2018 using nationwide administrative healthcare registries. Model predictors and outcome were pre-specified. Predictors were age, sex, education, use of antithrombotic, blood pressure-lowering, glucose-lowering, or lipid-lowering drugs, and a smoking proxy of smoking-cessation drug use or chronic obstructive pulmonary disease. Outcome was 5-year risk of first CVD event, a combination of ischaemic heart disease, heart failure, peripheral artery disease, stroke, or cardiovascular death. Predictions were computed using cause-specific Cox regression models. The final model fitted in the full data was internally-externally validated in each Danish Region. The model was well-calibrated in all regions. Area under the receiver operating characteristic curve (AUC) and Brier scores ranged from 76.3% to 79.6% and 3.3 to 4.4. The model was superior to an age-sex benchmark model with differences in AUC and Brier scores ranging from 1.2% to 1.5% and -0.02 to -0.03. Average predicted risks in each Danish municipality ranged from 2.8% to 5.9%. Predicted risks for a 66-year old ranged from 2.6% to 25.3%. Personalized predicted risks across ages 30-85 were presented in an online calculator (https://hjerteforeningen.shinyapps.io/cvd-risk-manuscript/)., Conclusion: A CVD risk prediction model based solely on nationwide administrative registry data provided accurate prediction of personal and population-level 5-year first CVD event risk in the Danish population. This may inform clinical and public health primary prevention efforts., (© The Author(s) 2021. Published by Oxford University Press on behalf of the European Society of Cardiology.)

A group of milk components that has shown potential to be predicted with milk spectra is milk minerals. Milk minerals are important for human health and cow health. Having an inexpensive and fast way to measure milk mineral concentrations would open doors for research, herd management, and selective breeding. The first aim of this study was to predict milk minerals with infrared milk spectra. Additionally, milk minerals were predicted with infrared-predicted fat, protein, and lactose content. The second aim was to perform a genetic analysis on infrared-predicted milk minerals, to identify QTL, and estimate variance components. For training and validating a multibreed prediction model for individual milk minerals, 264 Danish Jersey cows and 254 Danish Holstein cows were used. Partial least square regression prediction models were built for Ca, Cu, Fe, K, Mg, Mn, Na, P, Se, and Zn based on 80% of the cows, selected randomly. Prediction models were externally validated with 8 herds based on the remaining 20% of the cows. The prediction models were applied on a population of approximately 1,400 Danish Holstein cows with 5,600 infrared spectral records and 1,700 Danish Jersey cows with 7,200 infrared spectral records. Cows from this population had 50k imputed genotypes. Prediction accuracy was good for P and Ca, with external R 2 ≥ 0.80 and a relative prediction error of 5.4% for P and 6.3% for Ca. Prediction was moderately good for Na with an external R 2 of 0.63, and a relative error of 18.8%. Prediction accuracies of milk minerals based on infrared-predicted fat, protein, and lactose content were considerably lower than those based on the infrared milk spectra. This shows that the milk infrared spectrum contains valuable information on milk minerals, which is currently not used. Heritability for infrared-predicted Ca, Na, and P varied from low (0.13) to moderate (0.36). Several QTL for infrared-predicted milk minerals were observed that have been associated with gold standard milk minerals previously. In conclusion, this study has shown infrared milk spectra were good at predicting Ca, Na, and P in milk. Infrared-predicted Ca, Na, and P had low to moderate heritability estimates., (The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).)

Objective: School-age children with attention-deficit/hyperactivity disorder (ADHD) have reduced health-related quality of life (HRQoL), but it is unclear whether this is also true for preschool children. It is unknown whether parent training (PT) improves HRQoL. This study compared HRQoL in preschool children with ADHD with age-matched children from the general population; examined whether PT improves HRQoL; and tested if treatment-related changes in HRQoL were mediated by improvements in ADHD, parent efficacy, and family stress., Method: Parents of 164 children age 3-7 years with an ADHD diagnosis participated in a randomized controlled trial comparing the New Forest Parenting Programme and treatment as usual. Measures of HRQoL, ADHD, parent efficacy, and family stress were completed at baseline, posttreatment, and 36-week follow-up. Child baseline HRQoL was compared with 2 general population-based reference groups. PT effects were analyzed using linear models and mediation analyses., Results: Preschoolers with ADHD had lower HRQoL than the reference groups. The New Forest Parenting Programme, but not treatment as usual, was associated with improvement in psychosocial HRQoL at posttreatment (change 2.28, 95% CI [0.78, 3.77]) and at 36-week follow-up (change 2.05, 95% CI [0.56, 3.54]). This difference between treatment arms was not statistically significant. Parent efficacy and family stress scores at posttreatment significantly mediated improvements in HRQoL at 36-week follow-up; ADHD scores at posttreatment did not., Conclusion: ADHD negatively impacts HRQoL in early childhood. PT for ADHD has the potential to improve HRQoL independently of its effects on ADHD symptoms., Clinical Trial Registration Information: A Controlled Study of Parent Training in the Treatment of ADHD in Young Children (D'SNAPP); http://clinicaltrial.gov/; NCT01684644., (Copyright © 2020 American Academy of Child and Adolescent Psychiatry. Published by Elsevier Inc. All rights reserved.)

Background: Preventive primary care programs that aim to reduce morbidity and mortality from lifestyle-related diseases are often affected by low-to-moderate participation rates. Improving participation rates is essential for clinical effectiveness and cost-effectiveness. In 2016-2017, we conducted a pilot study (TOF pilot1) for a preventive primary care intervention (TOF is the Danish abbreviation for "Early Detection and Prevention"). Among 8814 invited patients, 3545 (40.22%) consented to participate, with the highest participation rates among women and patients with higher income, education, and employment., Objective: The aim of this study was to evaluate the effects of a revised invitation strategy on invitation comprehensibility, the overall participation rate, and participant demography. The new strategy specifically targeted men and patients of low educational attainment., Methods: This study was embedded in the second TOF pilot study (TOF pilot2, initiated in October 2018) that tested an adjusted intervention. The revised invitation strategy comprised a prenotification postcard and a new invitation that specifically targeted men and patients of low educational attainment. The new invitation was developed in a co-design process that included communication professionals and target-group representatives. The study sample consisted of 4633 patients aged between 29 and 59 years, who resided in one of two municipalities in the Region of Southern Denmark. Eligible patients were randomly assigned to one of four invitation groups. The control group (Group 1) received the original invitation used in TOF pilot1. The intervention groups received the original invitation and the prenotification postcard (Group 2), the new revised invitation and the prenotification postcard (Group 3), or the new invitation but no prenotification postcard (Group 4)., Results: Overall, 2171 (46.86%) patients consented to participate. Compared to the control group, participation rates increased significantly in all three intervention groups (all P<.001). Participation across the three intervention groups increased for women and men, as well as for patients with high and low educational attainment and high and low family income. The largest relative increase in participation rates occurred among men, patients with low educational attainment, and patients with low family income. No increase in participation was detected for unemployed patients or patients of non-Danish origin. Most participants found the original (813/987, 82.37%) and new (965/1133, 85.17%) invitations easy to understand with no significant difference (P=.08) in comprehensibility between invitations., Conclusions: The results suggest that participation in preventive primary care interventions can be greatly increased by implementing a co-design-based invitation strategy that includes prenotification postcards and targeted invitations. Although firm conclusions cannot be made from this study, the observed increased participation rates for men and patients of low socioeconomic status may be relevant in programs that aim to reduce social inequality in health., Trial Registration: ClinicalTrials.gov NCT03913585; https://clinicaltrials.gov/ct2/show/NCT03913585., (©Trine Thilsing, Lars Bruun Larsen, Anders Larrabee Sonderlund, Signe Skaarup Andreassen, Jeanette Reffstrup Christensen, Nanna Herning Svensson, Marie Dahl, Jens Sondergaard. Originally published in JMIR Public Health and Surveillance (http://publichealth.jmir.org), 10.03.2021.)

Researchers working with neural networks have historically focused on either non-spiking neurons tractable for running on computers or more biologically plausible spiking neurons typically requiring special hardware. However, in nature homogeneous networks of neurons do not exist. Instead, spiking and non-spiking neurons cooperate, each bringing a different set of advantages. A well-researched biological example of such a mixed network is a sensorimotor pathway, responsible for mapping sensory inputs to behavioral changes. This type of pathway is also well-researched in robotics where it is applied to achieve closed-loop operation of legged robots by adapting amplitude, frequency, and phase of the motor output. In this paper we investigate how spiking and non-spiking neurons can be combined to create a sensorimotor neuron pathway capable of shaping network output based on analog input. We propose sub-threshold operation of an existing spiking neuron model to create a non-spiking neuron able to interpret analog information and communicate with spiking neurons. The validity of this methodology is confirmed through a simulation of a closed-loop amplitude regulating network inspired by the internal feedback loops found in insects for posturing. Additionally, we show that non-spiking neurons can effectively manipulate post-synaptic spiking neurons in an event-based architecture. The ability to work with mixed networks provides an opportunity for researchers to investigate new network architectures for adaptive controllers, potentially improving locomotion strategies of legged robots., 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 Strohmer, Stagsted, Manoonpong and Larsen.)

Milk oligosaccharides are of high interest due to their bioactive properties. This study is the first to characterise milk oligosaccharides from native North European cattle breeds, as represented by 80 milk samples collected from eight native breeds originated from Norway (Norwegian Doela cattle and Norwegian Telemark cattle), Sweden (Swedish Mountain cattle), Denmark (Danish Red anno 1970), Iceland (Icelandic cattle), Lithuania (native Lithuanian Black and White) and Finland (Western Finncattle and Eastern Finncattle). Using high-performance liquid-chromatography chip/quadrupole time-of-flight mass-spectrometry, 18 unique monosaccharide compositions and a multitude of isomers were identified. No N-glycolylneuraminic acid was identified among these breeds. Western Finncattle milk was most abundant in neutral, acidic and fucosylated oligosaccharides. Further, Eastern Finncattle milk was significantly higher in acidic oligosaccharides and Icelandic cattle milk significantly higher in fucosylated oligosaccharides, compared to the mean. This study highlights specific native breeds of particular interest for future exploitation of milk oligosaccharides and breeding strategies., Competing Interests: Declaration of interest: none

Our objective was to determine the content of the bioactive protein osteopontin (OPN) in bovine milk and identify factors influencing its concentration. OPN is expressed in many tissues and body fluids, with by far the highest concentrations in milk. OPN plays a role in immunological and developmental processes and it has been associated with several milk production traits and lactation persistency in cows. In the present study, we report the development of an enzyme linked immunosorbent assay (ELISA) for measurement of OPN in bovine milk. The method was used to determine the concentration of OPN in milk from 661 individual Danish Holstein cows. The median OPN level was determined to 21.9 mg/l with a pronounced level of individual variation ranging from 0.4 mg/l to 67.8 mg/l. Breeding for increased OPN in cow's milk is of significant interest, however, the heritability of OPN in milk was found to be relatively low, with an estimated value of 0.19 in the current dataset. The variation explained by the herd was also found to be low suggesting that OPN levels are not affected by farm management or feeding. Interestingly, the concentration of OPN was found to increase with days in milk and to decrease with parity.

Purpose: To investigate the clinical impact of FDG-PET/CT for staging and treatment planning in high-risk primary breast cancer., Methods: Women with high-risk primary breast cancer were enrolled between September 2017 and August 2019 at Odense University Hospital, Denmark. Conventional mammography with/without MRI was performed before staging by FDG-PET/CT. We studied the accuracy of FDG-PET/CT for the detection of distant metastases, the effect on the change of treatment, and the prevalence of incidental findings. Biopsy and follow-up were used as a reference standard for the accuracy analysis., Results: Of 103 women, 24 (23%) were diagnosed with distant metastases by FDG-PET/CT. Among these, breast surgery was omitted in 18 and could have been spared in six. Another sixteen (16%) patients were upstaged to more advanced loco-regional disease, leading to more extensive radiotherapy. Sensitivity and specificity for diagnosing distant metastases were 1.00 (95% confidence interval: 0.86-1.00) and 0.95 (0.88-0.99), respectively. Twenty-nine incidental findings were detected in 24 women (23%), leading to further examinations in 22 and diagnosis of eight (8/22, 36%) synchronous diseases: cancer (n = 4), thyroiditis (n = 2), aorta aneurysm (n = 1), and meningioma (n = 1)., Conclusions: FDG-PET/CT had a substantial impact on staging and change of treatment in women with high-risk primary breast cancer, and further examination of incidental findings was considered clinically relevant. Our findings suggest that FDG-PET/CT should be considered for primary staging in high-risk primary breast cancer to improve treatment planning.

Background: Preventive health checks targeted at the at-risk population can be a way of preventing noncommunicable diseases. However, evidence on patient preferences for preventive health checks is limited, especially among patients with a high risk of noncommunicable diseases., Objective: To examine patient preferences for preventive health checks in Danish general practice, targeting persons at high risk of a noncommunicable disease., Methods: The method used in this study was a discrete choice experiment (DCE) with five attributes: assess, advice, agree, assist and arrange. The attributes were inspired by the 5A model for behaviour change counselling but was altered for the purpose of this study to grasp the entirety of the general practice-based intervention. Moreover, the attribute levels were defined to resemble daily clinical practice. The experimental design of the DCE was an efficient Bayesian main effects design and the results were analysed using a random utility theory framework., Results: A total of 148 patients completed the DCE. Patients at high risk of a noncommunicable disease have positive preferences for: giving brief explanations about own lifestyle, practicing shared decision-making with the general practitioner (GP), follow-up counselling with the GP after the preventive health check and scheduling a new appointment right after the preventive health check., Conclusions: The results provide Danish GPs with evidence on their patients' preferences towards preventive health checks which will enable the GPs to tailor these consultations. Moreover, the results suggest that pre-appointment measures, such as a health profile, may mediate a preference for more action-oriented attributes., Trial Registration: Registered at Clinical Trial Gov (Unique Protocol ID: TOFpilot2016, https://clinicaltrials.gov/ct2/show/NCT02797392?term=TOFpilot2016&rank=1). Prospectively registered on the 29th of April 2016., (© The Author(s) 2020. Published by Oxford University Press.)

High-value milk proteins, which can be obtained by optimized fractionation procedures, are ideal ingredients in many food applications. Thus, a simple and robust analytical method is required for the identification and quantification of these individual milk proteins. Here, we present a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method using multiple reaction monitoring (MRM) to simultaneously detect and measure target peptides of two major milk proteins, α-lactalbumin (α-LA) and β-casein (β-CN), in raw milk samples from 662 Danish Holstein cows. The MRM quantification of α-LA and β-CN was achieved with limit of detection (LOD) of 0.14 and 0.16 g/L, respectively and reproducibility of the assay <15%. By this newly established MRM-based method, the concentration of α-LA and β-CN in an individual cow's milk ranged from 0.5 to 1.9 (average 1.1) g/L, and from 7.5 to 23.4 (average 15) g/L, respectively. There was no significant effect of parity, whereas significantly increasing concentrations of α-LA and β-CN were observed through lactation (P < 0.001). This shows a considerable biological variation of these two ingredient milk proteins, providing potential varying outputs of fractionation in the dairy streams., (© 2020 The Authors.)

Artificial intelligence (AI) has the potential to increase quality and efficiency of breast cancer screening. Recent studies have provided comparative data on AI versus human performance in cancer detection with encouraging results, and commercially available AI systems are used worldwide as a clinical tool for mammography screening. There are, however, a number of methodological concerns in relation to the evaluation of AI systems. This review discusses these aspects as well as the opportunities and challenges of clinical validation and implementation in breast cancer screening practice.

Background: Recent evidence suggests the effectiveness of stepwise, targeted approaches for the prevention of lifestyle-related diseases with combinations of web-based and face-to-face interventions showing promising results., Objective: This paper reports on 1-year changes in health-risk behaviors, BMI, self-rated health, mental well-being, and risk of disease at 1-year follow-up after participation in a stepwise intervention that targeted persons at high risk of disease and persons with health-risk behavior. To this end, we distinguish between participants who took up the full intervention (web-based plus face-to-face) and those who received only the web-based intervention., Methods: The Early Detection and Prevention (Danish acronym: TOF) pilot study was conducted as a nonrandomized, 1-year follow-up intervention study in two municipalities in the Region of Southern Denmark. A total of 9400 citizens born between 1957 and 1986 (aged 29 to 60 years) were randomly sampled from participating general practitioner (GP) patient-list systems and were invited to take part in the study. Participants were subsequently stratified into risk groups based on their responses to a questionnaire on health-risk behavior and data from their GP's electronic patient record (EPR) system. All participants received a digital personal health profile with individualized information on current health-risk behavior and targeted advice on relevant health-risk behavior changes. In addition, patients at high risk of disease, as indicated by their digital health profile, were offered a targeted intervention at their GP. Patients who were not deemed at high risk of disease but who exhibited health-risk behaviors were offered a targeted intervention at their municipal health center (MHC). At 1-year follow-up, health-risk behaviors, self-rated health, BMI, and mental well-being were reassessed by questionnaire, and current information on diagnoses and medical treatment was retrieved from the EPRs., Results: Of 598 patients at high risk of disease or with health-risk behavior, 135 took up the targeted intervention at their GP or MHC and 463 received the personal health profile only. From baseline to 1-year follow-up, the number of patients with unhealthy eating habits decreased, mean mental well-being increased, and smoking prevalence decreased in patients who had received the digital personal health profile alone. Among patients who took up the targeted intervention, unhealthy eating habits and sedentary lifestyles decreased and significant reductions in mean BMI were observed. At 1-year follow up, no health-risk behaviors were detected among 17.4% of patients who at baseline had exhibited health-risk behaviors or high risk of disease., Conclusions: A stepwise targeted preventive approach using web-based and face-to-face elements may lead to favorable lifestyle changes. Specifically, a web-based approach may improve smoking and eating habits and mental well-being, whereas supplementary face-to-face interventions may be necessary to improve exercise habits and BMI., Trial Registration: ClinicalTrials.gov NCT02797392; https://clinicaltrials.gov/ct2/show/NCT02797392., International Registered Report Identifier (irrid): RR2-10.1186/s12875-018-0820-8., (©Trine Thilsing, Anders Larrabee Sonderlund, Jens Sondergaard, Nanna Herning Svensson, Jeanette Reffstrup Christensen, Janus Laust Thomsen, Niels Christian Hvidt, Lars Bruun Larsen. Originally published in JMIR Public Health and Surveillance (http://publichealth.jmir.org), 09.07.2020.)

During heat treatment of milk, β-lactoglobulin (β-LG) associates with the milk fat globule membrane (MFGM). The objective of this study was to examine different binding types that could be involved in this process. First, we tested the thiol-disulfide bond interchange between β-LG and MFGM by heating raw milk (87°C, 8 min) in the presence of different reagents capable of preventing this interaction, and then evaluated the presence of β-LG in resulting MFGM preparations by sodium dodecyl sulfate-PAGE. Contrary to commonly accepted theory, β-LG still associated with MFGM when milk was heated in the presence of 10 mM N-ethylmaleimide, dithiobis-nitrobenzoic acid, or dithioerythritol. This finding indicated that noncovalent binding could be involved in the interaction, and therefore these were studied next. Preventing noncovalent interactions by heating milk in the presence of 8 M urea (to inhibit formation of hydrogen bonds) or 2 M NaCl (to inhibit electrostatic and hydrophobic interactions) reduced the association of β-LG and MFGM. Inhibiting both hydrogen and disulfide bond formation by addition of 8 M urea and 10 mM dithioerythritol or inhibiting hydrophobic interactions with 0.2% sodium dodecyl sulfate completely prevented the association. In contrast to the simple thiol-disulfide interaction model, the results suggest a more complex understanding of the interactions between β-LG and MFGM during heating of milk. This indicates that disulfide formation between β-LG and proteins in the MFGM is not required for the association, but that hydrophobic interactions and hydrogen bonding may be crucial. This novel insight into β-LG and MFGM association is in contrast to the current literature and requires further study., (Copyright © 2020 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Neural signals for locomotion are influenced both by the neural network architecture and sensory inputs coordinating and adapting the gait to the environment. Adaptation relies on the ability to change amplitude, frequency, and phase of the signals within the sensorimotor loop in response to external stimuli. However, in order to experiment with closed-loop control, we first need a better understanding of the dynamics of the system and how adaptation works. Based on insights from biology, we developed a spiking neural network capable of continuously changing amplitude, frequency, and phase online. The resulting network is deployed on a hexapod robot in order to observe the walking behavior. The morphology and parameters of the network results in a tripod gait, demonstrating that a design without afferent feedback is sufficient to maintain a stable gait. This is comparable to results from biology showing that deafferented samples exhibit a tripod-like gait and adds to the evidence for a meaningful role of network topology in locomotion. Further, this work enables research into the role of sensory feedback and high-level control signals in the adaptation of gait types. A better understanding of the neural control of locomotion relates back to biology where it can provide evidence for theories that are currently not testable on live insects., (Copyright © 2020 Strohmer, Manoonpong and Larsen.)

The effect of the addition of caseinomacropeptide (CMP) or desialylated CMP on the heat-induced denaturation and aggregation of whey proteins was investigated in the pH range 3 to 7 after heating at 80°C for 30 min. The rate and temperature of denaturation, the extent of aggregation, and the changes in secondary structure of the whey proteins heated in presence of CMP or desialylated CMP were measured. The sialic acid bound to CMP favored the denaturation and aggregation of whey proteins when the whey proteins were oppositely charged to CMP at pH 4. A transition occurred at pH 6, below which the removal of sialic acid enhanced the stabilizing properties of CMP against the denaturation and aggregation of the whey proteins. At pH >6, the interactions between desialylated CMP and the whey proteins led to more extensive denaturation and aggregation. Sialic acid bound to CMP influenced the denaturation and aggregation behavior of whey proteins in a pH-dependent manner, and this should be considered in future studies on the heat stability of such systems containing CMP., (Copyright © 2020 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)