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Laura Cartwright1, Marie Sønnegaard Poulsen2, Hanne Mørck Nielsen3, Giulio Pojana4, Lisbeth E Knudsen2, Margaret Saunders1, Erik Rytting2,51Bristol Initiative for Research of Child Health (BIRCH), Biophysics Research Unit, St Michael's Hospital, UH Bristol NHS Foundation Trust, Bristol, UK; 2University of Copenhagen, Faculty of Health Sciences, Department of Public Health, 3University of Copenhagen, Faculty of Pharmaceutical Sciences, Department of Pharmaceutics and Analytical Chemistry, Copenhagen, Denmark; 4Department of Environmental Sciences, Informatics and Statistics, University Ca' Foscari Venice, Venice, Italy; 5Department of Obstetrics and Gynecology, University of Texas Medical Branch, Galveston, Texas, USABackground: Advances in biomedical nanotechnology raise hopes in patient populations but may also raise questions regarding biodistribution and biocompatibility, especially during pregnancy. Special consideration must be given to the placenta as a biological barrier because a pregnant woman's exposure to nanoparticles could have significant effects on the fetus developing in the womb. Therefore, the purpose of this study is to optimize an in vitro model for characterizing the transport of nanoparticles across human placental trophoblast cells.Methods: The growth of BeWo (clone b30) human placental choriocarcinoma cells for nanoparticle transport studies was characterized in terms of optimized Transwell® insert type and pore size, the investigation of barrier properties by transmission electron microscopy, tight junction staining, transepithelial electrical resistance, and fluorescein sodium transport. Following the determination of nontoxic concentrations of fluorescent polystyrene nanoparticles, the cellular uptake and transport of 50 nm and 100 nm diameter particles was measured using the in vitro BeWo cell model.Results: Particle size measurements, fluorescence readings, and confocal microscopy indicated both cellular uptake of the fluorescent polystyrene nanoparticles and the transcellular transport of these particles from the apical (maternal) to the basolateral (fetal) compartment. Over the course of 24 hours, the apparent permeability across BeWo cells grown on polycarbonate membranes (3.0 µm pore size) was four times higher for the 50 nm particles compared with the 100 nm particles.Conclusion: The BeWo cell line has been optimized and shown to be a valid in vitro model for studying the transplacental transport of nanoparticles. Fluorescent polystyrene nanoparticle transport was size-dependent, as smaller particles reached the basal (fetal) compartment at a higher rate.Keywords: nanoparticles, placenta, BeWo cells, transport, model optimization, nanotoxicology

Purpose: To investigate the association between irradiated breast volume and grade 2-3 breast induration three years after radiotherapy in the phase III Danish Breast Cancer Group HYPO trial randomizing patients ≥ 41 years to whole breast irradiation (WBI) with 40 Gy/15fr versus 50 Gy/25fr., Methods: Treatment plans were available for all Danish patients. Associations between frequency of induration and irradiated volume, age, smoking status, and boost were assessed by logistic regression. A sequential boost was given to patients < 50 years or in case of a narrow (<2 mm) resection margin., Results: RT plans from 1,333 patients were analyzed with 178 (13 %) having grade 2-3 induration. 1135 patients had only WBI. For this group, induration was correlated with irradiated breast volume for patients ≥ 65 years (n = 343, 10 %/22 % for small/large irradiated volumes, p = 0.005) but not for patients aged 50-64 years (n = 792, 11 % for both small and large volumes, p = 0.82). Smoking doubled the frequency irrespective of irradiated volume and age. All patients < 50 years (n = 156) had a boost. A volume effect was found for this group (5 %/21 % induration for small/large volume, p = 0.002). 42 patients ≥ 50 years had a boost and 14 (33 %) had grade 2-3 induration, however, with a p-value > 0.05 due to the few numbers of patients., Conclusion: A relationship between irradiated breast volume and 3-year frequency of breast induration was found for patients ≥ 65 years, whilst not for patients aged 50-64 years. Smoking doubled the risk of induration irrespective of volume and age. A dose-induration relationship was seen for boost patients < 50 years., 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 Author(s). Published by Elsevier B.V. All rights reserved.)

Milk production and overall dairy farm economics depend on rearing dairy heifers. This study investigated the presence of a genotype by environment interaction in Holstein (HOL), Nordic Red dairy cattle (RDC), and their F 1 crossbreeds (HOL × RDC) when provided different feed rations. The aim of our study was to assess how different energy concentrations in feed rations affect growth, BCS, feed intake, and feed efficiency in the 3 groups during the prepubertal period. The 3 breed groups were randomly allocated to receive either a standard or a low-energy feed ration. Holstein heifers exhibited reduced growth and a lower BCS when they were fed the low-energy feed ration. In contrast, the RDC heifers demonstrated similar growth rates with the different feed rations and maintained similar BCS irrespective of feed energy concentration. The HOL × RDC crossbred heifers performed as an intermediate between the HOL and RDC groups. Significant differences were observed in DMI and energy intake in the HOL and HOL × RDC groups depending on feed ration treatment. The RDC heifers had similar feed intake irrespective of treatment. There were no significant differences in the feed conversion ratio among breeds and feed treatments. These results indicate the presence of a genotype by environment interaction in prepubertal HOL and RDC heifers in response to differences in feed ration treatment. Due to the influence of prepubertal growth on future milk production, reproduction, and health status, it is important to be aware of breed-specific requirements during the prepubertal period, particularly in mixed breed and crossbred groups, to optimize growth rates and production potential., (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/).)

Native mucus is heterogeneous, displays high inter-individual variation and is prone to changes during harvesting and storage. To overcome the lack of reproducibility and availability of native mucus, commercially available purified mucins, porcine gastric mucin (PGM) and mucin from bovine submaxillary gland (BSM), have been widely used. However, the question is to which extent the choice of mucin matters in studies of their interaction with polymers as their composition, structure and hence physicochemical properties differ. Accordingly, the interactions between PGM or BSM with two widely used polymers in drug delivery, polyethylene oxide and chitosan, was studied with orthogonal methods: turbidity, dynamic light scattering, and quartz crystal microbalance with dissipation monitoring. Polymer binding and adsorption to the two commercially available and purified mucins, PGM and BSM, is different depending on the mucin type. PEO, known to interact weakly with mucin, only displayed limited interaction with both mucins as confirmed by all employed methods. In contrast, chitosan was able to bind to both PGM and BSM. Interestingly, the results suggest that chitosan interacts with BSM to a greater extent than with PGM indicating that the choice of mucin, PGM or BSM, can affect the outcome of studies of mucin interactions with polymers., (© 2024. The Author(s).)

Black soldier fly (BSF; Hermetia illucens) is a promising insect species for food and feed production as its larvae can convert different organic waste to high-value protein. Selective breeding is one way to optimize production, but the potential of breeding is only starting to be explored and not yet utilized for BSF. To assist in monitoring a captive population and implementing a breeding program, genomics tools are imperative. We conducted whole genome sequencing of two captive populations separated by geographical distance - Denmark (DK) and Texas, USA (TX). Various population genetics analyses revealed a moderate genetic differentiation between two populations. Moreover, we observed higher inbreeding in the DK population, and the detection of a subpopulation within DK population aligned well with the recent foundation of the DK population from two captive populations. Additionally, we generated gene ontology annotation and variants annotation for wider potential applications. Our findings establish a robust marker set for research in population genetics, facilitating the monitoring of inbreeding and laying the groundwork for practical breeding programs for BSF., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Dr. Tomberlin has a significant financial interest in EVO Conversion Systems LLC. This conflict of interest is managed by a plan submitted to and approved by Texas A&M University and Texas A&M AgriLife., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: There is a burgeoning interest in using insects as a sustainable source of food and feed, particularly by capitalising on various waste materials and by-products that are typically considered of low value. Enhancing the commercial production of insects can be achieved through two main approaches: optimising environmental conditions and implementing selective breeding strategies. In order to successfully target desirable traits through selective breeding, having a thorough understanding of the genetic parameters pertaining to those traits is essential. In this study, a full-sib half-sib mating design was used to estimate variance components and heritabilities for larval size and survival at day seven of development, development time and survival from egg to adult, and to estimate correlations between these traits, within an outbred population of house flies (Musca domestica), using high-throughput phenotyping for data collection., Results: The results revealed low to intermediate heritabilities and positive genetic correlations between all traits except development time and survival to day seven of development and from egg to adulthood. Surprisingly, larval size at day seven exhibited a comparatively low heritability (0.10) in contrast to development time (0.25), a trait that is believed to have a stronger association with overall fitness. A decline in family numbers resulting from low mating success and high overall mortality reduced the amount of available data which resulted in large standard errors for the estimated parameters. Environmental factors made a substantial contribution to the phenotypic variation, which was overall high for all traits., Conclusions: There is potential for genetic improvement in all studied traits and estimates of genetic correlations indicate a partly shared genetic architecture among the traits. All estimates have large standard errors. Implementing high-throughput phenotyping is imperative for the estimation of genetic parameters in fast developing insects, and facilitates age synchronisation, which is vital in a breeding population. In spite of endeavours to minimise non-genetic sources of variation, all traits demonstrated substantial influences from environmental components. This emphasises the necessity of thorough attention to the experimental design before breeding is initiated in insect populations., (© 2024. The Author(s).)

Apolipoprotein E (APOE) genetic variants are most notably known for their divergent impact on the risk of developing Alzheimer's disease. While APOE genotype has been consistently shown to modulate lipid metabolism in a variety of cellular contexts, the effect of APOE alleles on the lipidome in hepatocytes is unknown. In this study, we investigated the contribution of APOE alleles to lipidomic profiles of donor-derived primary human hepatocytes from 77 subjects. Lipidomic data obtained by liquid chromatography-mass spectrometry were analyzed across ε2/ε3, ε3/ε3, and ε3/ε4 genotypes to reveal how APOE modulates lipid relative levels over age and between groups. Hepatic APOE concentration, measured by ELISA, was assessed for correlation with lipid abundance in subjects grouped as per APOE genotype and sex. APOE genotype-specific differential lipidomic signatures associated with age for multiple lipid classes but did not differ between sexes. Compared to ε2/ε3, ε3/ε4 hepatocytes had higher abundance of acylcarnitines (AC) and acylphosphatidylglycerol (AcylPG) as a class, as well as higher medium and long-chain ACs, AcylPG, phosphatidylglycerol (PG), bis(monoacylglycerol)phosphate (BMP), monoacylglycerol (MG) and diacylglycerol (DG) species. The ε3/ε4 hepatocytes also exhibited a higher abundance of medium and long-chain ACs compared to the ε3/ε3 hepatocytes. Only in the ε3/ε4 hepatocytes, APOE concentration was lower and showed a negative correlation with BMP levels, specifically in females. APOE genotype dictates a differential lipidome in primary human hepatocytes. The lipids involved suggest mitochondrial dysfunction with accompanying alterations in neutral lipid storage, reflective of a general disturbance of free fatty acid metabolism in human hepatocytes with the ε4 allele., Competing Interests: Conflict of 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 Inc. All rights reserved.)

Cancer in pregnancy is rare, and most physicians lack knowledge in handling pregnant cancer patients. This review summarises the present knowledge on this condition. In the Netherlands, an Advisory Board on Cancer in Pregnancy was established in 2012. The board supports Dutch physicians' decisions in the management of pregnant patients with cancer. In 2021 the International Advisory Board on Cancer in Pregnancy was established, and in continuation, the Danish Advisory Board on Cancer in Pregnancy (DABCIP) has now been founded. DABCIP consists of 22 members from 13 different medical disciplines., (Published under Open Access CC-BY-NC-BD 4.0. https://creativecommons.org/licenses/by-nc-nd/4.0/.)

Antimicrobial peptides (AMPs) are promising alternatives to antibiotics for treatment of antimicrobial resistant (AMR) bacterial infections. However, their narrow therapeutic window due to in vivo toxicity and limited stability hampers their clinical use. Here, we evaluated encapsulation of two amphiphilic AMPs, SAAP-148 and snake cathelicidin Ab-Cath, into oleyl-modified hyaluronic acid (OL-HA) nanogels to improve their selectivity index. The AMP-loaded OL-HA nanogels ranged 181-206 nm in size with a PDI of 0.2, highly negative surface charge (-47 to -48 mV) and moderate encapsulation efficiency (53-63%). The AMP-loaded OL-HA nanogels displayed similar activity in vitro as AMP solutions against AMR Staphylococcus aureus and Acinetobacter baumannii, with a dose-dependent effect over time. Importantly, the AMP-loaded OL-HA nanogels showed decreased cytotoxicity towards human erythrocytes and primary skin fibroblast, thereby improving the selectivity index of SAAP-148 and Ab-Cath by 2- and 16.8-fold, respectively. Particularly, the selectivity of Ab-Cath-loaded OL-HA nanogels has great clinical potential, with an index that reached ≥ 300 for S. aureus and ≥ 3000 for A. baumannii. These findings indicate that OL-HA nanogels are a promising drug delivery system to reduce the cytotoxicity of AMPs without substantially affecting their antimicrobial activity, thereby increasing their selectivity index and potential as therapeutics to combat AMR bacterial infections., 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 © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

The cell-penetrating peptide penetratin and its analogues shuffle and penetramax have been used as carrier peptides for oral delivery of therapeutic peptides such as insulin. Their mechanism of action for this purpose is not fully understood but is believed to depend on the interactions of the peptide with the cell membrane. In the present study, peptide-liposome interactions were investigated using advanced biophysical techniques including small-angle neutron scattering and fluorescence lifetime imaging microscopy. Liposomes were used as a model system for the cell membrane. All the investigated carrier peptides induced liposome clustering at a specific peptide/lipid ratio. However, distinctively different types of membrane interactions were observed, as the liposome clustering was irreversible for penetratin, but fully or partly reversible for shuffle and penetramax, respectively. All three peptides were found to adsorb to the surface of the lipid bilayers, while only shuffle and penetramax led to shape deformation of the liposomes. Importantly, the peptide interactions did not disrupt the liposomes under any of the investigated conditions, which is advantageous for their application in drug delivery. This detailed insight on peptide-membrane interactions is important for understanding the mechanism of peptide-based excipients and the influence of peptide sequence modifications., 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 © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Metastatic triple-negative breast cancer (mTNBC) is an aggressive subtype of breast cancer with poor survival. Currently, the literature lacks comprehensive real-world evidence on locally recurrent and mTNBC patients. To validate the optimal treatment for patients with mTNBC, real-world evidence in combination with data from clinical trials must be evaluated as complementary., Objectives: The objective of the study is to examine outcomes and treatment patterns of patients with advanced triple-negative breast cancer (TNBC) utilizing real-world data of patients from all oncology sites across Denmark., Design: This is a retrospective, non-interventional, multi-site, population-based observational study conducted across all oncology departments in Denmark., Methods: We included all women diagnosed with metastatic or locally recurrent TNBC from January 1, 2017, to December 31, 2019, using the national Danish Breast Cancer Group database. The primary endpoints were overall survival (OS) and progression-free survival (PFS) in the first to third treatment line., Results: The study included 243 women diagnosed with metastatic or recurrent TNBC. The median OS (mOS) was 11.6 months after the first line of treatment, 6.5 months after the second line, and 6.5 months after the third line. De novo mTNBC was associated with shorter OS (mOS: 8.3 vs 14.2 months), and those with a relapse within 18 months of primary diagnosis had shorter OS than those with a relapse after 18 months (mOS: 10.0 vs 18.2). In the first line, taxane was the preferred choice of treatment for patients with de novo mTNBC, whereas capecitabine was preferred for patients with recurrent TNBC., Conclusions: This real-world, nationwide study demonstrated poor OS among patients with metastatic or recurrent TNBC, with a mOS of 11.6 months (95% CI, 9.9-17.3). Patients who presented with de novo mTNBC or who had a relapse of their breast cancer within 18 months of primary diagnosis had shorter OS., Registration: The study was registered and approved by the Danish Capital Regions research overview (P-2021-605)., Competing Interests: The author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: TB: Institutional grants from Danish Cancer Society, Neye-fonden, Roche, Novartis, Samsung Bioepis, Pfizer, AstraZeneca, Merck Eisai, and Venture Oncology Personal; Invited Speaker: Pfizer; and Advisory Board: Merck. MJ: Institutional grants from Samsung Bioepis, Nanostring Technologies, and Venture Oncology. AK: Institutional grants from Roche and Venture Oncology Personal and Advisory Board: Novartis, AstraZeneca, MDS, Roche, Pfizer, and Eli Lilly Danmark A/S. Other authors had no conflict of interest to declare., (© The Author(s) 2023.)

Introduction: The Danish Medicines Council recommends that patients with estrogen receptor and human epidermal growth factor receptor 2-negative, programmed death-ligand 1 (PD-L1)-positive advanced breast cancer receive atezolizumab in combination with nab-paclitaxel. The approval was largely based on results from Impassion130 that showed a beneficial progression-free survival (PFS) and overall survival (OS) in PD-L1-positive patients who received atezolizumab and nab-paclitaxel., Methods: We conducted a retrospective, population-based study that included patients who received atezolizumab for advanced breast cancer from October 2019 to September 2022. The primary endpoints were PFS and OS., Results: This study included 74 advanced breast cancer patients. Their median age was 54.5 years, and 21 (28.4%) of the patients had de novo advanced disease. Most patients received first-line treatment with atezolizumab (83.8%). The median PFS was 6.0 months (95% confidence interval (CI): 4.7-8.4 months) and the median OS was 14.3 months (95% CI: 9.9-22.2 months). A total of 48 patients received atezolizumab and nab-paclitaxel in accordance with guidelines from the Danish Medicines Council., Conclusions: This real-world study expectedly showed numerically lower survival outcomes than the phase III trial Impassion130, but met the standards of efficacy set by real-world studies in other countries. A need exists for increased attention to the criteria for receiving atezolizumab., Funding: none., Trial Registration: The study was approved by the Oncological Committee of the DBCG, the Research Overview of the Capital (P-2022-828) and the Centre for Health of the Capital Region (R-22060674)., (Articles published in the DMJ are “open access”. This means that the articles are distributed under the terms of the Creative Commons Attribution Non-commercial License, which permits any non-commercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.)

Competing Interests: Declaration of competing interest The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results

Background: In commercial pig production, reduction of harmful social behavioural traits, such as ear manipulation and tail biting, is of major interest. Moreover, farmers prefer animals that are easy to handle. The aim of this experiment was to determine whether selection on social breeding values (SBV) for growth rate in purebred pigs affects behaviour in a weighing crate, lesions from ear manipulation, and tail biting of their crossbred progeny. Data were collected on crossbred F1 pigs allocated to 274 pens, which were progeny of purebred Landrace sows and Yorkshire boars from a DanBred nucleus herd., Results: Behaviour in the weighing crate scored on a three-level scale showed that groups of pigs with high SBV for growth rate were significantly calmer than groups of pigs with low SBV (P < 0.027). When the mean SBV in the group increased by 1 unit, the proportion of pigs that obtained a calmer score level was increased by 14%. A significant (p = 0.04), favourable effect of SBV was found on both the number of pigs with ear lesions in the group and the mean number of ear lesions per pig. For a 1 unit increase in mean SBV, the mean number of lesions per pig decreased by 0.06 from a mean of 0.98. Individual severity of ear lesions conditional upon the number of ear lesions was also significantly affected (p = 0.05) by the mean SBV in the group. In groups for which the mean SBV increased by 1 unit, the proportion of pigs that were observed with a lower severity score was increased by 20% on a three-level scale. Most pigs received no tail biting injuries and no effect of SBV was observed on the tail injury score., Conclusions: After 7 weeks in the finisher unit, crossbred progeny with high SBV were calmer in the weighing crate and had fewer ear lesions. These results indicate that selection of purebred parents for SBV for growth rate will increase welfare in their crossbred progeny by decreasing the number of ear lesions and making them easier to handle., (© 2023. The Author(s).)

Background: The APOEε4-promoted risk of Alzheimer's disease (AD) is lower in Black/African-Americans (B/AAs), compared to non-Hispanic whites (NHWs). Previous studies reported lower plasma apolipoprotein E (apoE) levels in NHW APOEε4-carriers compared to non-carriers, and low plasma apoE levels were directly associated with an increased risk of AD and all dementia. We further showed that APOEε3/ε3 AD patients exhibited reduced plasma apoE dimers compared to corresponding control subjects. Whether plasma apoE levels and apoE dimer formation differ between races/ethnicities and therefore may help explain AD risk racial disparity remains to be elucidated., Methods: Using mass spectrometry, we determined total plasma apoE and apoE isoform levels in a cohort of B/AAs (n = 58) and NHWs (n = 67) including subjects with normal cognition (B/AA: n = 25, NHW: n = 28), mild cognitive impairment (MCI) (B/AA: n = 24, NHW: n = 24), or AD dementia (B/AA: n = 9, NHW: n = 15). Additionally, we used non-reducing western blot analysis to assess the distribution of plasma apoE into monomers/disulfide-linked dimers. Plasma total apoE, apoE isoform levels, and % apoE monomers/dimers were assessed for correlations with cognition, cerebrospinal fluid (CSF) AD biomarkers, sTREM2, neurofilament light protein (NfL), and plasma lipids., Results: Plasma apoE was predominantly monomeric in both racial groups and the monomer/dimer distribution was not affected by disease status, or correlated with CSF AD biomarkers, but associated with plasma lipids. Plasma total apoE levels were not related to disease status and only in the NHW subjects we observed lower plasma apoE levels in the APOEε4/ε4-carriers. Total plasma apoE levels were 13% higher in B/AA compared to NHW APOEε4/ε4 subjects and associated with plasma high-density lipoprotein (HDL) in NHW subjects but with low-density lipoprotein levels (LDL) in the B/AA subjects. Higher plasma apoE4 levels, exclusively in APOEε3/ε4 B/AA subjects, were linked to higher plasma total cholesterol and LDL levels. In the controls, NHWs and B/AAs exhibited opposite associations between plasma apoE and CSF t-tau., Conclusions: The previously reported lower APOEε4-promoted risk of AD in B/AA subjects may be associated with differences in plasma apoE levels and lipoprotein association. Whether differences in plasma apoE levels between races/ethnicities result from altered APOEε4 expression or turnover, needs further elucidation., (© 2023. The Author(s).)

Cell-penetrating peptides (CPPs), such as penetratin, are often investigated as drug delivery vectors and incorporating d-amino acids, rather than the natural l-forms, to enhance proteolytic stability could improve their delivery efficiency. The present study aimed to compare membrane association, cellular uptake, and delivery capacity for all-l and all-d enantiomers of penetratin (PEN) by using different cell models and cargos. The enantiomers displayed widely different distribution patterns in the examined cell models, and in Caco-2 cells, quenchable membrane binding was evident for d-PEN in addition to vesicular intracellular localization for both enantiomers. The uptake of insulin in Caco-2 cells was equally mediated by the two enantiomers, and while l-PEN did not increase the transepithelial permeation of any of the investigated cargo peptides, d-PEN increased the transepithelial delivery of vancomycin five-fold and approximately four-fold for insulin at an extracellular apical pH of 6.5. Overall, while d-PEN was associated with the plasma membrane to a larger extent and was superior in mediating the transepithelial delivery of hydrophilic peptide cargoes compared to l-PEN across Caco-2 epithelium, no enhanced delivery of the hydrophobic cyclosporin was observed, and intracellular insulin uptake was induced to a similar degree by the two enantiomers.

Introduction: At the Alzheimer's Association's APOE and Immunity virtual conference, held in October 2021, leading neuroscience experts shared recent research advances on and inspiring insights into the various roles that both the apolipoprotein E gene (APOE) and facets of immunity play in neurodegenerative diseases, including Alzheimer's disease and other dementias., Methods: The meeting brought together more than 1200 registered attendees from 62 different countries, representing the realms of academia and industry., Results: During the 4-day meeting, presenters illuminated aspects of the cross-talk between APOE and immunity, with a focus on the roles of microglia, triggering receptor expressed on myeloid cells 2 (TREM2), and components of inflammation (e.g., tumor necrosis factor α [TNFα])., Discussion: This manuscript emphasizes the importance of diversity in current and future research and presents an integrated view of innate immune functions in Alzheimer's disease as well as related promising directions in drug development., (© 2023 The Authors. Alzheimer's & Dementia published by Wiley Periodicals LLC on behalf of Alzheimer's Association.)

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.

Oromucosal administration is an attractive non-invasive route. However, drug absorption is challenged by salivary flow and the mucosa being a significant permeability barrier. The aim of this study was to design and investigate a multi-layered nanofiber-on-foam-on-film (NFF) drug delivery system with unique properties and based on polysaccharides combined as i) mucoadhesive chitosan-based nanofibers, ii) a peptide loaded hydroxypropyl methylcellulose foam, and iii) a saliva-repelling backing film based on ethylcellulose. NFF displays optimal mechanical properties shown by dynamic mechanical analysis, and biocompatibility demonstrated after exposure to a TR146 cell monolayer. Chitosan-based nanofibers provided the NFF with improved mucoadhesion compared to that of the foam alone. After 1 h, >80 % of the peptide desmopressin was released from the NFF. Ex vivo permeation studies across porcine buccal mucosa indicated that NFF improved the permeation of desmopressin compared to a commercial freeze-dried tablet. The findings demonstrate the potential of the NFF as a biocompatible drug delivery system., Competing Interests: Declaration of competing interest Mai Bay Stie, Heidi Öblom, Jette Jacobsen, Jukka Rantanen, Hanne M. Nielsen, Natalja Genina are inventors of the NFF as covered by the submitted patent application PCT/EP2022/059128 entitled “Multilayered patch”., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The inherent low oral bioavailability of therapeutic peptides can be enhanced by the cell-penetrating peptide penetratin and its analogues shuffle and penetramax applied as carriers for delivery of insulin. In this study, the objective was to gain mechanistic insights on the effect of the carrier peptide stereochemistry on their interactions with insulin and on insulin delivery. Insulin-carrier peptide interactions were investigated using small-angle X-ray scattering and cryogenic transmission electron microscopy, while the insulin and peptide stability and transepithelial insulin permeation were evaluated in the Caco-2 cell culture model along with the carrier peptide-induced effects on epithelial integrity and cellular metabolic activity. Interestingly, the insulin transepithelial permeation was influenced by the degree of insulin-carrier peptide complexation and depended on the stereochemistry of penetramax but not of penetratin and shuffle. The l-form of the peptides initially decreased the epithelial integrity comparable to that induced by the d-peptides, suggesting a comparable mechanism of action. The immediate decrease was reversible during exposure of the Caco-2 epithelium to the l-peptides but not during exposure to the d-peptides, likely a result of their higher stability. Overall, exploration of the stereochemistry showed to be an interesting strategy for carrier peptide-mediated insulin delivery.

Amyloid aggregation is associated with many diseases and may also occur in therapeutic protein formulations. Addition of co-solutes is a key strategy to modulate the stability of proteins in pharmaceutical formulations and select inhibitors for drug design in the context of diseases. However, the heterogeneous nature of this multicomponent system in terms of structures and mechanisms poses a number of challenges for the analysis of the chemical reaction. Using insulin as protein system and polysorbate 80 as co-solute, we combine a spatially resolved fluorescence approach with single molecule microscopy and machine learning methods to kinetically disentangle the different contributions from multiple species within a single aggregation experiment. We link the presence of interfaces to the degree of heterogeneity of the aggregation kinetics and retrieve the rate constants and underlying mechanisms for single aggregation events. Importantly, we report that the mechanism of inhibition of the self-assembly process depends on the details of the growth pathways of otherwise macroscopically identical species. This information can only be accessed by the analysis of single aggregate events, suggesting our method as a general tool for a comprehensive physicochemical characterization of self-assembly reactions.

Plasma apolipoprotein E levels were previously associated with the risk of developing Alzheimer's disease (AD), levels of cerebrospinal fluid AD biomarkers, cognition and imaging brain measures. Outside the brain, the liver is the primary source of apoE and liver transplantation studies have demonstrated that liver-derived apoE does not cross the blood-brain-barrier. How hepatic apoE may be implicated in behavioral and cognitive performance is not clear. In the current study, we behaviorally tested FRGN mice with humanized liver harboring the ε3/ε3 genotype (E3-human liver (HL)) and compared their behavioral and cognitive performance with that of age-matched ε3/ε3 targeted replacement (E3-TR) mice, the latter produces human apoE3 throughout the body whereas the E3-HL mice endogenously produce human apoE3 only in the liver. We also compared the liver weights and plasma apoE levels, and assessed whether plasma apoE levels were correlated with behavioral or cognitive measures in both models. E3-HL were more active but performed cognitively worse than E3-TR mice. E3-HL mice moved more in the open field containing objects, showed higher activity levels in the Y maze, showed higher activity levels during the baseline period in the fear conditioning test than E3-TR mice, and swam faster than E3-TR mice during training to locate the visible platform in the water maze. However, E3-HL mice showed reduced spatial memory retention in the water maze and reduced fear learning and contextual and cued fear memory than E3-TR mice. Liver weights were greater in E3-HL than E3-TR mice and sex-dependent only in the latter model. Plasma apoE3 levels were similar to those found in humans and comparable in female and male E3-TR mice but higher in female E3-HL mice. Finally, we found correlations between plasma apoE levels and behavioral and cognitive measures which were predominantly model-dependent. Our study demonstrates mouse-model dependent associations between plasma apoE levels, behavior and cognition in an 'AD-neutral' setting and suggests that a humanized liver might be sufficient to induce mouse behavioral and cognitive phenotypes., (© 2023. The Author(s).)

Chronic wound infections colonized by bacteria are becoming more difficult to treat with current antibiotics due to the development of antimicrobial resistance (AMR) as well as biofilm and persister cell formation. Synthetic antibacterial and antibiofilm peptide (SAAP)-148 is an excellent alternative for treatment of such infections but suffers from limitations related to its cationic peptidic nature and thus instability and possible cytotoxicity, resulting in a narrow therapeutic window. Here, we evaluated SAAP-148 encapsulation in nanogels composed of octenyl succinic anhydride (OSA)-modified hyaluronic acid (HA) to circumvent these limitations. SAAP-148 was efficiently (>98%) encapsulated with high drug loading (23%), resulting in monodispersed anionic OSA-HA nanogels with sizes ranging 204-253 nm. Nanogel lyophilization in presence of polyvinyl alcohol maintained their sizes and morphology. SAAP-148 was sustainedly released from lyophilized nanogels (37-41% in 72 h) upon reconstitution. Lyophilized SAAP-148-loaded nanogels showed similar antimicrobial activity as SAAP-148 against planktonic and biofilm-residing AMR Staphylococcus aureus and Acinetobacter baumannii . Importantly, formulated SAAP-148 showed reduced cytotoxicity against human erythrocytes, primary human skin fibroblasts and human keratinocytes. Additionally, lyophilized SAAP-148-loaded nanogels eradicated AMR S. aureus and A. baumannii colonizing a 3D human epidermal model, without inducing any cytotoxicity in contrast to SAAP-148. These findings indicate that OSA-HA nanogels increase SAAP-148's therapeutic potential for treatment of skin wound infections.

Purpose: On the basis of low risk of local recurrence in elderly patients with breast cancer after conservative surgery followed by whole breast irradiation (WBI), the Danish Breast Cancer Group initiated the noninferiority external-beam partial breast irradiation (PBI) trial (ClinicalTrials.gov identifier: NCT00892814). We hypothesized that PBI was noninferior to WBI regarding breast induration., Methods: Patients operated with breast conservation for relatively low-risk breast cancer were randomly assigned to WBI versus PBI, and all had 40 Gy/15 fractions. The primary end point was 3-year grade 2-3 breast induration., Results: In total, 865 evaluable patients (434 WBI and 431 PBI) were enrolled between 2009 and 2016. Median follow-up was 5.0 years (morbidity) and 7.6 years (locoregional recurrence). The 3-year rate of induration was 9.7% for WBI and 5.1% for PBI ( P = .014). Large breast size was significantly associated with induration with a 3-year incidence of 13% (WBI) and 6% (PBI) for large-breasted patients versus 6% (WBI) and 5% (PBI) for small-breasted patients. PBI showed no increased risk of dyspigmentation, telangiectasia, edema, or pain, and patient satisfaction was high. Letrozole and smoking did not increase the risk of radiation-associated morbidity. Sixteen patients had a locoregional recurrence (six WBI and 10 PBI; P = .28), 20 patients had a contralateral breast cancer, and eight patients had distant failure (five WBI and three PBI). A nonbreast second cancer was detected in 73 patients (8.4%), and there was no difference between groups., Conclusion: External-beam PBI for patients with low-risk breast cancer was noninferior to WBI in terms of breast induration. Large breast size was a risk factor for radiation-associated induration. Few recurrences were detected and unrelated to PBI.

Drug delivery systems (DDS) for oral delivery of peptide drugs contain excipients that facilitate and enhance absorption. However, little knowledge exists on how DDS excipients such as permeation enhancers interact with the gastrointestinal mucus barrier. This study aimed to investigate interactions of the permeation enhancer sodium 8-[(2-hydroxybenzoyl)amino]octanoate (SNAC) with ex vivo porcine intestinal mucus (PIM), ex vivo porcine gastric mucus (PGM), as well as with in vitro biosimilar mucus (BM) by profiling their physical and barrier properties upon exposure to SNAC. Bulk mucus permeability studies using the peptides cyclosporine A and vancomycin, ovalbumin as a model protein, as well as fluorescein-isothiocyanate dextrans (FDs) of different molecular weights and different surface charges were conducted in parallel to mucus retention force studies using a texture analyzer, rheological studies, cryo-scanning electron microscopy (cryo-SEM), and single particle tracking of fluorescence-labelled nanoparticles to investigate the effects of the SNAC-mucus interaction. The exposure of SNAC to PIM increased the mucus retention force, storage modulus, viscosity, increased nanoparticle confinement within PIM as well as decreased the permeation of cyclosporine A and ovalbumin through PIM. Surprisingly, the viscosity of PGM and the permeation of cyclosporine A and ovalbumin through PGM was unaffected by the presence of SNAC, thus the effect of SNAC depended on the regional site that mucus was collected from. In the absence of SNAC, the permeation of different molecular weight and differently charged FDs through PIM was comparable to that through BM. However, while bulk permeation of neither of the FDs through PIM was affected by SNAC, the presence of SNAC decreased the permeation of FD4 and increased the permeation of FD150 kDa through BM. Additionally, and in contrast to observations in PIM, nanoparticle confinement within BM remained unaffected by the presence of SNAC. In conclusion, the present study showed that SNAC altered the physical and barrier properties of PIM, but not of PGM. The effects of SNAC in PIM were not observed in the BM in vitro model. Altogether, the study highlights the need for further understanding how permeation enhancers influence the mucus barrier and illustrates that the selected mucus model for such studies should be chosen with care., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Purpose: The relation between breast induration grade 2-3 at 3 years after radiation therapy and irradiated breast volume was investigated for patients in the Danish Breast Cancer Group (DBCG) Partial Breast Irradiation (PBI) trial. METHODS Treatment plan data was obtained from the Danish radiotherapy plan database. Dosimetric parameters for breast and organs at risk were determined. Breast induration data was obtained from the DBCG database. The volume of the whole breast (CTVp&#95;breast) treated to various dose levels was determined for treatment plans in both arms. Logistic regression was used to assess the frequency of induration on breast volume irradiated to ≥40 Gy. RESULTS PBI and WBI was given to 433 and 432 patients, respectively. Median and interquartile ranges (IQR) for CTVp&#95;breast were 710 mL (467-963 mL; PBI) and 666 mL (443-1012 mL; WBI) (p = 0.98). Median and IQR for CTVp&#95;breast treated to ≥40 Gy was 24.9% (18.6-32.6%; PBI) and 59.8% (53.6-68.5%; WBI). Grade 2-3 induration was observed in 5% (PBI) and 10% (WBI) of the patients. A dose-response relationship was established between irradiated breast volume and frequency of breast induration. From the model, 5% and 10% risks of breast induration were observed for ≥40 Gy delivered to CTVp&#95;breast volumes of 177 mL (95%CI, 94-260 mL) and 426 mL (95%CI, 286-567 mL), respectively. CONCLUSION The frequency of breast induration increased significantly with increasing irradiated breast volume, strongly favouring small volumes and PBI. Thus, treated breast volume - not the breast size itself - is the risk factor for induration. This is the first report directly linking the 40 Gy irradiated breast volume to breast induration., (Copyright © 2022 Elsevier B.V. All rights reserved.)

ApoE is the major lipid and cholesterol carrier in the CNS. There are three major human polymorphisms, apoE2, apoE3, and apoE4, and the genetic expression of APOE4 is one of the most influential risk factors for the development of late-onset Alzheimer's disease (AD). Neuroinflammation has become the third hallmark of AD, together with Amyloid-β plaques and neurofibrillary tangles of hyperphosphorylated aggregated tau protein. This review aims to broadly and extensively describe the differential aspects concerning apoE. Starting from the evolution of apoE to how APOE's single-nucleotide polymorphisms affect its structure, function, and involvement during health and disease. This review reflects on how APOE's polymorphisms impact critical aspects of AD pathology, such as the neuroinflammatory response, particularly the effect of APOE on astrocytic and microglial function and microglial dynamics, synaptic function, amyloid-β load, tau pathology, autophagy, and cell-cell communication. We discuss influential factors affecting AD pathology combined with the APOE genotype, such as sex, age, diet, physical exercise, current therapies and clinical trials in the AD field. The impact of the APOE genotype in other neurodegenerative diseases characterized by overt inflammation, e.g., alpha- synucleinopathies and Parkinson's disease, traumatic brain injury, stroke, amyotrophic lateral sclerosis, and multiple sclerosis, is also addressed. Therefore, this review gathers the most relevant findings related to the APOE genotype up to date and its implications on AD and CNS pathologies to provide a deeper understanding of the knowledge in the APOE field., (© 2022. The Author(s).)

Background: Low levels of plasma apolipoprotein E (apoE) and presence of the APOE ε4 allele are associated with an increased risk of Alzheimer's disease (AD). Although the increased risk of AD in APOE ε4-carriers is well-established, the protein levels have received limited attention., Methods: We here report the total plasma apoE and apoE isoform levels at baseline from a longitudinally (24 months) followed cohort including controls (n = 39), patients with stable amnestic mild cognitive impairment during 24 months follow up (MCI-MCI, n = 30), patients with amnestic MCI (aMCI) that during follow-up were clinically diagnosed with AD with dementia (ADD) (MCI-ADD, n = 28), and patients with AD with dementia (ADD) at baseline (ADD, n = 28). We furthermore assessed associations between plasma apoE levels with cerebrospinal fluid (CSF) AD biomarkers and α-synuclein, as well as both CSF and plasma neurofilament light chain (NfL), YKL-40 and kallikrein 6., Results: Irrespective of clinical diagnosis, the highest versus the lowest apoE levels were found in APOE ε2/ε3 versus APOE ε4/ε4 subjects, with the most prominent differences exhibited in females. Total plasma apoE levels were 32% and 21% higher in the controls versus MCI-ADD and ADD patients, respectively. Interestingly, MCI-ADD patients exhibited a 30% reduction in plasma apoE compared to MCI-MCI patients. This decrease appeared to be associated with brain amyloid-β (Aβ 42 ) pathology regardless of disease status as assessed using the Amyloid, Tau, and Neurodegeneration (A/T/N) classification. In addition to the association between low plasma apoE and low levels of CSF Aβ 42 , lower apoE levels were also related to higher levels of CSF total tau (t-tau) and tau phosphorylated at Threonine 181 residue (p-tau) and NfL as well as a worse performance on the mini-mental-state-examination. In MCI-ADD patients, low levels of plasma apoE were associated with higher levels of CSF α-synuclein and kallikrein 6. No significant correlations between plasma apoE and the astrocytic inflammatory marker YKL40 were observed., Conclusions: Our results demonstrate important associations between low plasma apoE levels, Aβ pathology, and progression from aMCI to a clinical ADD diagnosis., (© 2022. The Author(s).)

Liver-generated plasma apolipoprotein E (apoE) does not enter the brain but nonetheless correlates with Alzheimer's disease (AD) risk and AD biomarker levels. Carriers of APOEε4, the strongest genetic AD risk factor, exhibit lower plasma apoE and altered brain integrity already at mid-life versus non-APOEε4 carriers. Whether altered plasma liver-derived apoE or specifically an APOEε4 liver phenotype promotes neurodegeneration is unknown. Here we investigated the brains of Fah-/-, Rag2-/-, Il2rg-/- mice on the Non-Obese Diabetic (NOD) background (FRGN) with humanized-livers of an AD risk-associated APOE ε4/ε4 versus an APOE ε2/ε3 genotype. Reduced endogenous mouse apoE levels in the brains of APOE ε4/ε4 liver mice were accompanied by various changes in markers of synaptic integrity, neuroinflammation and insulin signaling. Plasma apoE4 levels were associated with unfavorable changes in several of the assessed markers. These results propose a previously unexplored role of the liver in the APOEε4-associated risk of neurodegenerative disease., (© 2022. The Author(s).)

Polyelectrolyte complexes (PECs) have been used as the matrix of solid foams for drug delivery. This study aimed at investigating the effect of graphene oxide (GO) and the composition of excipients in chitosan/alginate-based buccal foams on the clotrimazole release and antifungal activities. The investigation has been focused on the interactions of the drug with excipients in the foams, and the changes of ionization degree upon exposure to various media are discussed. The solid foams were prepared by mixing the excipients and clotrimazole via probe sonication, followed by a freeze-drying method. The pH values of the formulations were measured during the foam preparation process to estimate the ionization degree of clotrimazole and the other excipients. The foam matrix was the PECs between the cationic chitosan and anionic alginate. The mechanical strength of clotrimazole-loaded foams was lower than that of drug-free foams due to the positively charged clotrimazole interacting with the anionic alginate and interfering the PECs between chitosan and alginate. Addition of GO in the clotrimazole-loaded matrix made the foams mechanically stronger and contributed to a faster release of clotrimazole from the buccal foams by disrupting the electrostatic interactions between alginate and clotrimazole. However, addition of 1 wt% GO in the formulations didn't affect the antifungal activity of clotrimazole-loaded foams significantly. A lower amount GO in the formulation may be required for enhancing the antifungal effect, which should be further investigated in future., (Copyright © 2022 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background: In patients with atopic dermatitis (AD), Staphylococcus aureus frequently colonizes lesions and is hypothesized to be linked to disease severity and progression. Treatments that reduce S. aureus colonization without significantly affecting the skin commensal microbiota are needed., Methods and Findings: In this study, we tested ATx201 (niclosamide), a small molecule, on its efficacy to reduce S. aureus and propensity to evolve resistance in vitro. Various cutaneous formulations were then tested in a superficial skin infection model. Finally, a Phase 2 randomized, double-blind and placebo-controlled trial was performed to investigate the impact of ATx201 OINTMENT 2% on S. aureus colonization and skin microbiome composition in patients with mild-to-severe AD (EudraCT:2016-003501-33). ATx201 has a narrow minimal inhibitory concentration distribution (.125-.5 μg/ml) consistent with its mode of action - targeting the proton motive force effectively stopping cell growth. In murine models, ATx201 can effectively treat superficial skin infections of methicillin-resistant S. aureus. In a Phase 2 trial in patients with mild-to-severe AD (N = 36), twice-daily treatment with ATx201 OINTMENT 2% effectively reduces S. aureus colonization in quantitative colony forming unit (CFU) analysis (primary endpoint: 94.4% active vs. 38.9% vehicle success rate, p = .0016) and increases the Shannon diversity of the skin microbiome at day 7 significantly compared to vehicle., Conclusion: These results suggest that ATx201 could become a new treatment modality as a decolonizing agent., (© 2022 The Authors. Clinical and Translational Medicine published by John Wiley & Sons Australia, Ltd on behalf of Shanghai Institute of Clinical Bioinformatics.)

Background: Between 1982 and 1990 the Danish Breast Cancer Cooperative Group (DBCG) conducted a randomized trial in high-risk pre- and postmenopausal (<70 years) breast cancer patients comparing mastectomy plus adjuvant systemic therapy alone versus the same treatment plus postoperative irradiation., Aim: To present a comprehensive analysis of the complete DBCG 82bc study with a 30-year long-term follow-up of the cancer therapeutic effect and survival, together with an additional focus on the potential long-term life-threatening morbidity related to cardiac irradiation and/or the risk of secondary cancer induction., Methods: A total of 3083 patients with pathological stage II and stage III breast cancer were after mastectomy randomly assigned to receive adjuvant systemic therapy and postoperative irradiation to the chestwall and regional lymph nodes (1538 pts), or adjuvant systemic therapy alone (1545 pts). Pre- and menopausal patients (DBCG 82b) received 8-9 cycles of CMF with an interval of 4 weeks, whereas postmenopausal patients (DBCG 82c) received tamoxifen 30 mg daily for one year. The median follow-up time was 34 years. The primary endpoints were loco-regional recurrence (LRR) and overall mortality, and the secondary endpoints were distant metastasis, breast cancer mortality, and irradiation related late morbidity., Results: Overall the 30-year cumulative incidence of loco-regional recurrence was 9% in irradiated patients versus 37% in non-irradiated patients who received adjuvant systemic therapy alone (HR: 0.21 [95% cfl 0.18-0.26]). Distant metastasis probability at 30 years was 49% in irradiated patients compared to 60% in non-irradiated (HR: 0.77 [0.70-0.84]). Consequently, these figures resulted in a reduced breast cancer mortality: 56% vs 67% (HR: 0.75 [0.69-0.82], and overall mortality (81% vs 86% at 30 years (p < 0.0001), HR: 0.83 [0.77-0.90] in favor of irradiation. Radiotherapy did not result in any significant excess death of other courses, such as ischemic heart disease, HR: 0.82 [0.58-1.18]; nor secondary lung cancer HR: 1.44 [0.92-2.24], or other non-cancer related death HR: 1.15 [0.92-1.45]., Conclusion: The study definitely demonstrate that optimal long-term treatment benefit of high-risk breast cancer can only be achieved if both loco-regional and systemic tumor control are aimed for. Therefore, radiotherapy has an important role in the multidisciplinary treatment of breast cancer. The PMRT treatment did not result in excess ischemic heart damage, nor in other non-breast cancer related death., Competing Interests: Conflict of interest The authors declare that none of them have any conflicts on interest in relation to the present publication., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Current organic pig-breeding programs use pigs from conventional breeding populations. However, there are considerable differences between conventional and organic production systems. This simulation study aims to evaluate how the organic pig sector could benefit from having an independent breeding program. Two organic pig-breeding programs were simulated: one used sires from a conventional breeding population (conventional sires), and the other used sires from an organic breeding population (organic sires). For maintaining the breeding population, the conventional population used a conventional breeding goal, whereas the organic population used an organic breeding goal. Four breeding goals were simulated: one conventional breeding goal, and three organic breeding goals. When conventional sires were used, genetic gain in the organic population followed the conventional breeding goal, even when an organic breeding goal was used to select conventional sires. When organic sires were used, genetic gain followed the organic breeding goal. From an economic point of view, using conventional sires for breeding organic pigs is best, but only if there are no genotype-by-environment interactions. However, these results show that from a biological standpoint, using conventional sires biologically adapts organic pigs for a conventional production system.

The sublingual mucosa is an attractive route for drug delivery, although challenged by a continuous flow of saliva that leads to a loss of drug by swallowing. It is of great benefit that drugs absorbed across the sublingual mucosa avoid exposure to the harsh environment of the gastro-intestinal lumen; this is especially beneficial for drugs of low physicochemical stability such as therapeutic peptides. In this study, a two-layered hybrid drug delivery system was developed for the sublingual delivery of the therapeutic peptide desmopressin. It consisted of peptide-loaded mucoadhesive electrospun chitosan/polyethylene oxide-based nanofibers (mean diameter of 183 ± 20 nm) and a saliva-repelling backing film to promote unidirectional release towards the mucosa. Desmopressin was released from the nanofiber-based hybrid system (approximately 80% of the loaded peptide was released within 45 min) in a unidirectional manner in vitro. Importantly, the nanofiber-film hybrid system protected the peptide from wash-out, as demonstrated in an ex vivo flow retention model with porcine sublingual mucosal tissue. Approximately 90% of the loaded desmopressin was retained at the surface of the ex vivo porcine sublingual mucosa after 15 min of exposure to flow rates representing salivary flow.

This chapter describes the use of cell-penetrating peptides (CPPs) as carriers for transepithelial delivery of therapeutic peptides. Assessment of transepithelial peptide permeation and the mechanisms of action that permeability enhancing drug carriers exert on the epithelium requires subtle sample preparation and analysis by orthogonal methods. Here, the preparation and use of CPP-insulin physical mixture samples including the quantification of insulin by enzyme-linked immunosorbent assay (ELISA) is described. In addition, effects of CPPs on the epithelium and its barrier properties immediately upon exposure and after a recovery period are evaluated by epithelial cell viability, transepithelial electrical resistance, immunostaining of the tight junction associated zonula occludens (ZO-1) protein, and actin cytoskeleton staining., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Variations in symptom development among breast cancer (BC) survivors are understudied. We examined: (Q1) Symptom trajectories of pain, fatigue, insomnia, breast, and arm symptoms in BC survivors, (Q2) possible patterns or cluster-like associations between trajectory classification of different symptoms, and (Q3) characteristics of survivors assigned to high-burden symptom trajectories., Material and Methods: Participants were 968 women (mean age = 59.6 years) treated for early-stage BC and followed across a three-year postoperative period. As part of routine follow-up procedures, patients reported symptom burden and functioning levels at each hospital visit using the European Organisation for Research and Treatment of Cancer (EORTC) Quality of Life Questionnaire (QLQ-C30) and the BC-specific module (QLQ-BR-23). Growth mixture modeling (GMM) analysis was used to differentiate potential subgroups of individuals with similar longitudinal symptom patterns, i.e., symptom trajectories (Q1). With this approach, groups experiencing persistent, highly distressing cancer- and treatment-related late effects (LEs) may be identified. Latent class analysis (LCA) was used for Q2 and logistic regression analysis for Q3., Results: GMM identified two relatively parallel trajectories across the tested symptoms: The majority of the sample exhibited a low-burden symptom trajectory (74.4-89.2%) and a minority by a high-burden symptom trajectory (10.8-25.6%). LCA revealed that approximately one in five women (18.8%) were likely to be members of the high-burden symptom trajectory across all tested symptoms. In addition to a high probability of being burdened over time across multiple symptoms, these women were also characterized by poorer self-reported physical and social functioning., Conclusion: A substantial minority followed a high-burden symptom trajectory for several years following BC treatment. Associations were found in trajectory classification across symptoms, indicating that cancer-related LEs appear in clusters of multiple concurrent symptoms.

Delivery of therapeutic peptides upon oral administration is highly desired and investigations report that the cell-penetrating peptide (CPP) penetratin and its analogues shuffle and penetramax show potential as carriers to enhance insulin delivery. Exploring this, the specific aim of the present study was to understand the impact that their complexation with a lipidated or non-lipidated therapeutic cargo would have on the delivery, to evaluate the effect of differences in membrane interactions in vitro and in vivo, as well as to deduce the mode of action leading to enhanced delivery. Fundamental biophysical aspects were studied by a range of orthogonal methods. Transepithelial permeation of therapeutic peptide was evaluated using the Caco-2 cell culture model supplemented with epithelial integrity measurements, real-time assessment of the carrier peptide effects on cell viability and on mode of action. Pharmacokinetic and pharmacodynamic (PK/PD) parameters were evaluated following intestinal administration to rats and tissue effects were investigated by histology. The biophysical studies revealed complexation of insulin with shuffle and penetramax, but not with penetratin. This corresponded to enhanced transepithelial permeation of insulin, but not of lipidated insulin, when in physical mixture with shuffle or penetramax. The addition of shuffle and penetramax was associated with a lowering of Caco-2 cell monolayer integrity and viability, where the lowering of cell viability was immediate, but reversible. Insulin delivery in rats was enhanced by shuffle and penetramax and accompanied by a 10-20-fold decrease in blood glucose with immediate effect on the intestinal mucosa. In conclusion, shuffle and penetramax, but not penetratin, demonstrated to be potential candidates as carriers for transmucosal delivery of insulin upon oral administration, and their effect depended on association with both cargo and cell membrane. Interestingly, the present study provides novel mechanistic insight that peptide carrier-induced cargo permeation points towards enhancement via the paracellular route in the tight epithelium. This is different from the anticipated belief being that it is the cell-penetrating capability that facilitate transepithelial cargo permeation via a transcellular route., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Recalcitrant respiratory tract infections caused by bacteria have emerged as one of the greatest health challenges worldwide. Aerosolized antimicrobial therapy is becoming increasingly attractive to combat such infections, as it allows targeted delivery of high drug concentrations to the infected organ while limiting systemic exposure. However, successful aerosolized antimicrobial therapy is still challenged by the diverse biological barriers in infected lungs. Nanoparticle-mediated pulmonary drug delivery is gaining increasing attention as a means to overcome the biological barriers and accomplish site-specific drug delivery by controlling release of the loaded drug(s) at the target site. With the aim to summarize emerging efforts in combating respiratory tract infections by using nanoparticle-mediated pulmonary delivery strategies, this review provides a brief introduction to the bacterial infection-related pulmonary diseases and the biological barriers for effective treatment of recalcitrant respiratory tract infections. This is followed by a summary of recent advances in design of inhalable nanoparticle-based drug delivery systems that overcome the biological barriers and increase drug bioavailability. Finally, challenges for the translation from exploratory laboratory research to clinical application are also discussed and potential solutions proposed.

National and international across-population selection is often recommended and fairly common in the current breeding practice of dairy cattle, with the primary aims to increase genetic gain and genetic variability. The aim of this study was to test the hypothesis that the strategy of truncation selection of sires across populations [i.e., competitive gene flow strategy (CGF)] may not necessarily maximize genetic gain in the long term in the presence of genotype-by-environment interaction (G×E). Two alternative strategies used to be compared with CGF were forced gene flow (FGF) strategies, with 10 or 50% of domestic dams forced to be mated with foreign sires (FGF10%, FGF50%). Two equal-size populations (N dams = 1,000) that were selected for the same breeding goal trait (h 2 = 0.3) under G×E correlation (r g ) of either 0.9 or 0.8 were simulated to test these 3 different strategies. Each population first experienced either 5 or 20 differentiation generations (G d ), then 15 migration generations. Discrete generations were simulated for simplicity. Each population performed a within-population conventional breeding program during differentiation generations and the 3 across-population sire selection strategies based on joint genomic prediction during migration generations. The 4 G d&#95; r g combinations defined 4 different levels of differentiation degree between the 2 populations at the start of migration. The true rate of inbreeding over the last 10 migration generations in each scenario was constrained at 0.01 to provide a fair basis for comparison of genetic gain across scenarios. Results showed that CGF maximized the genetic gain after 15 migration generations in 5&#95;0.9 combination only, the case of the lowest differentiation degree, with a superiority of 0.4% (0.04 genetic SD units) over the suboptimal strategy. While in 5&#95;0.8, 20&#95;0.9, and 20&#95;0.8 combinations, 2 FGF strategies had a superiority in genetic gain of 2.3 to 12.5% (0.21-1.07 genetic SD units) over CGF after 15 migration generations, especially FGF50%. The superiority of FGF strategies over CGF was that they alleviated inbreeding, introduced new genetic variance in the early migration period, and improved accuracy in the entire migration period. Therefore, we concluded that CGF does not necessarily maximize the genetic gain of across-population genomic breeding programs given moderate G×E. The across-population selection strategy remains to be optimized to maximize genetic gain., (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/).)

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.

Within ecophysiological and genetic studies on insects, morphological and physiological traits are commonly assessed and phenotypes are typically obtained from manual measurements on numerous individuals. Manual observations are, however, time consuming, can introduce observer bias and are prone to human error. Here, we contrast results obtained from manual assessment of larval size and thermal tolerance traits in black soldier flies ( Hermetia illucens ) and houseflies ( Musca domestica ) that have been acclimated under three different temperature regimes with those obtained automatically using an image analysis software (Noldus EthoVision XT). We found that (i) larval size estimates of both species, obtained by manual weighing or by using the software, were highly correlated, (ii) measures of heat and cold tolerance using manual and automated approaches provided qualitatively similar results, and (iii) by using the software we obtained quantifiable information on stress responses and acclimation effects of potentially higher ecological relevance than the endpoint traits that are typically assessed when manual assessments are used. Based on these findings, we argue that automated assessment of insect stress responses and largescale phenotyping of morphological traits such as size will provide new opportunities within many disciplines where accurate and largescale phenotyping of insects is required.

Background: Average daily gain (ADG) in pigs is affected by both direct and social genetic effects (SGE). However, selection for SGE in purebreds has not conclusively been shown to improve ADG in crossbreds, and it is unknown whether SGE in purebreds are equal to those in crossbreds. Moreover, SGE may reflect dominance related behaviour, which is affected by the variation in body weight within a group. Therefore, we hypothesized that (a) there is a positive effect of parent average SGE estimated in purebred pigs on phenotypic ADG in crossbred offspring, and (b) there is an interaction between SGE on ADG and standard deviation in starting weight of pigs within the group. We also hypothesized that (c) social genetic variance for ADG exists in crossbred pigs, and (d) there is a favourable genetic correlation between SGE on ADG in purebred and crossbred pigs., Results: We found a statistically significant interaction between the standard deviation in starting weight and SGE within groups, and conditioning on the mean standard deviation in starting weight, we found a favourable regression coefficient (0.37 ± 0.21) of ADG in crossbreds on SGE in purebreds. Variances for SGE were small in both Landrace (L) and Yorkshire (Y), and higher for SGE in both the dam and sire component of crossbred YL. The genetic correlations between SGE in purebreds and the dam or sire component of SGE in crossbreds were also favourable (0.52 ± 0.48 and 0.34 ± 0.42, respectively), although not significantly different from 0., Conclusions: We confirmed that there is a positive effect of SGE estimated using purebred information on phenotypic ADG in crossbreds, and that the largest effect is achieved when the within-group variation in starting weight is small. Our results indicate that social genetic variance in crossbreds exists and that there is a favourable genetic correlation between social genetic effects in purebreds and crossbreds. Collectively, our results indicate that selection for SGE on ADG in purebreds in a nucleus farm environment with little competition for resources can improve ADG in crossbreds in a commercial environment.

Delivering macromolecular drugs, e.g. peptides, to the systemic circulation by oral administration is challenging due to their degradation in the gastrointestinal tract and low transmucosal permeation. In this study, the concept of an oral delivery device utilizing an elastomeric material is presented with the potential of increasing the absorption of peptides, e.g. insulin. Absorption enhancement in the intestine is proposed as a result of self-unfolding of a polydimethylsiloxane foil upon release from enteric coated capsules. A pH-sensitive polymer coating prevents capsule disintegration until arrival in the small intestine where complete unfolding of the elastomeric foil ensures close contact with the intestinal mucosa. Foils with close-packed hexagonal compartments for optimal drug loading are produced by casting against a deep-etched silicon master. Complete unfolding of the foil upon capsule disintegration is verified in vitro and the insulin release profile of the final delivery device confirms insulin protection at gastric pH. In vivo performance is evaluated with the outcome of quantifiable plasma insulin concentrations in all rats receiving duodenal administration of the novel delivery device. By taking advantage of elastomeric material properties for drug delivery, this approach might serve as inspiration for further development of commercially viable biocompatible devices for oral delivery of macromolecules., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: The thioredoxin-interacting protein (TXNIP) is involved in cellular metabolism and cell proliferation, and recently, deficient expression of TXNIP has been associated with progression and poor outcome for cancer patients., Objectives: To assess TXNIP expression and function in malignant T cells from cutaneous T-cell lymphoma (CTCL)., Methods: CTCL-derived malignant (MyLa2059, PB2B) and non-malignant (MyLa1850) cell lines were analysed by Western blotting and qPCR for TXNIP expression. Subsequently, the malignant CTCL cell lines were treated with GSK126 - an inhibitor of enhancer of zeste homolog 2 (EZH2) methyltransferase activity or assessed by bisulphite sequencing for TXNIP promoter methylation. Methylation was also assessed with the demethylating agent 5-azacytidine (5AZA). Finally, TXNIP was overexpressed in the malignant PB2B cell line via plasmid transduction, and the effect of TXNIP was further analysed by flow cytometry., Results: We report on low expression of TXNIP protein in all cell lines representing different subtypes and stages of CTCL when compared to non-malignant T cells. Epigenetic silencing and other mechanisms were involved in the repression of TXNIP whereas forced expression of TXNIP strongly inhibited proliferation of malignant T cells., Conclusions: Epigenetic silencing and other as yet unknown mechanisms repress TXNIP expression in malignant T cells. As forced expression of TXNIP inhibits malignant proliferation, we propose that TXNIP is a putative tumour suppressor in CTCL., (© 2020 S. Karger AG, Basel.)

During the last decade, the use of systematic crossbreeding in dairy cattle herds has increased in several countries of the world. The aim of this study was to estimate the effect of breed proportion and heterosis on milk production traits and udder health traits in dairy cattle. The study was based on records on milk yield (MY), protein yield (PY), fat yield (FY), somatic cell score (SCS), and mastitis (MAST) from 73,695 first-lactation dairy cows in 130 Danish herds applying systematic crossbreeding programs. Around 45% of the cows were crosses between Danish Holstein (DH), Danish Red (DR), or Danish Jersey (DJ), and the remaining were purebred DH, DR, or DJ. The statistical model included the fixed effects of herd-year, calving month, and calving age and an effect representing the lactation status of the cow. In addition, the model included a regression on calving interval from first to second lactation, a regression on the proportion of DH, DR, and DJ genes, and a regression on the degree of heterozygosity between DH and DR, DH and DJ, and DR and DJ. Random effects were the genetic effect of the cow and a residual. The effect of breed proportions was estimated relatively to DH. For MY, a pure DR yielded 461 kg milk less than DH, whereas a pure DJ yielded 2,259 kg milk less than a pure DH. Compared with DH, PY was 41.7 kg less for DJ, whereas PY for DR was 4.0 kg less than for DH. For FY, a DR yielded 10.6 kg less than DH, whereas there was no significant effect of breed proportion between DJ and DH. A DR cow had lower SCS (0.13) than DH, whereas DJ had higher SCS (0.14) than DH. There was no significant effect of breed proportion on MAST between the 3 breeds. Heterosis was significant in all combinations of breeds for MY, FY, and PY. Heterosis for crosses between DH and DR was 257 kg (3.2%), 11.9 kg (3.2%), and 8.9 kg (3.2%) for MY, PY, and FY, respectively. Corresponding figures for crosses between DH and DJ were 314 kg (4.4%), 14.3 kg (4.4%), and 10.4 kg (4.0%), whereas heterosis between DR and DJ was 462 kg (6.7%), 19.6 kg (6.7%), and 13.9 kg (5.4%) for MY, PY, and FY, respectively. Heterosis was only significant for SCS in the crosses between DH and DR. Heterosis effects for MAST were nonsignificant for all the crosses. The results obtained in this study demonstrate that in first lactation cows, there is a positive effect of heterosis on milk production traits, but limited effect on udder health traits., (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/).)

Background: Altered cerebral glucose metabolism, especially prominent in APOE ɛ4 carriers, occurs years prior to symptoms in Alzheimer's disease (AD). We recently found an association between a higher ratio of plasma apolipoprotein E4 (apoE4) over apoE3, and cerebral glucose hypometabolism in cognitively healthy APOE ɛ3/ɛ4 subjects. Plasma apoE does not cross the blood-brain barrier, hence we speculate that apoE is linked to peripheral glucose metabolism which is known to affect glucose metabolism in the brain., Objective: Explore potential associations between levels of plasma insulin and glucose with previously acquired plasma apoE, cerebral metabolic rate of glucose (CMRgl), gray matter volume, and neuropsychological test scores., Methods: Plasma insulin and glucose levels were determined by ELISA and a glucose oxidase assay whereas apoE levels were earlier quantified by mass-spectrometry in 128 cognitively healthy APOE ɛ3/ɛ4 subjects. Twenty-five study subjects had previously undergone FDG-PET and structural MRI., Results: Lower plasma apoE3 associated with higher plasma glucose but not insulin in male subjects and subjects with a body mass index above 25. Negative correlations were found between plasma glucose and CMRgl in the left prefrontal and bilateral occipital regions. These associations may have functional implications since glucose levels in turn were negatively associated with neuropsychological test scores., Conclusion: Plasma apoE3 but not apoE4 may be involved in insulin-independent processes governing plasma glucose levels. Higher plasma glucose, which negatively affects brain glucose metabolism, was associated with lower plasma apoE levels in APOE ɛ3/ɛ4 subjects. High plasma glucose and low apoE levels may be a hazardous combination leading to an increased risk of AD.