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Antibody phage-display technology identifies antibody-antigen interactions through multiple panning rounds, but traditional screening gives no information on enrichment or diversity throughout the process. This results in the loss of valuable binders. Next Generation Sequencing can overcome this problem. We introduce a high accuracy long-read sequencing method based on the recent Oxford Nanopore Technologies (ONT) Q20 + chemistry in combination with dual unique molecular identifiers (UMIs) and an optimized bioinformatic analysis pipeline to monitor the selections. We identified binders from two single-domain antibody libraries selected against a model protein. Traditional colony-picking was compared with our ONT-UMI method. ONT-UMI enabled monitoring of diversity and enrichment before and after each selection round. By combining phage antibody selections with ONT-UMIs, deep mining of output selections is possible. The approach provides an alternative to traditional screening, enabling diversity quantification after each selection round and rare binder recovery, even when the dominating binder was > 99% abundant. Moreover, it can give insights on binding motifs for further affinity maturation and specificity optimizations. Our results demonstrate a platform for future data guided selection strategies.

Bispecific antibodies (bsAbs) have attracted significant attention due to their dual binding activity, which permits simultaneous targeting of antigens and synergistic binding effects beyond what can be obtained even with combinations of conventional monospecific antibodies. Despite the tremendous therapeutic potential, the design and construction of bsAbs are often hampered by practical issues arising from the increased structural complexity as compared to conventional monospecific antibodies. The issues are diverse in nature, spanning from decreased biophysical stability from fusion of exogenous antigen-binding domains to antibody chain mispairing leading to formation of antibody-related impurities that are very difficult to remove. The added complexity requires judicious design considerations as well as extensive molecular engineering to ensure formation of high quality bsAbs with the intended mode of action and favorable drug-like qualities. In this review, we highlight and summarize some of the key considerations in design of bsAbs as well as state-of-the-art engineering principles that can be applied in efficient construction of bsAbs with diverse molecular formats.

Antibodies are attractive therapeutic candidates due to their ability to bind cognate antigens with high affinity and specificity. Still, the underlying molecular rules governing the antibody-antigen interface remain poorly understood, making in silico antibody design inherently difficult and keeping the discovery and design of novel antibodies a costly and laborious process. This study investigates the characteristics of antibody-antigen binding interfaces through a computational analysis of more than 850,000 atom-atom contacts from the largest reported set of antibody-antigen complexes with 1833 nonredundant, experimentally determined structures. The analysis compares binding characteristics of conventional antibodies and single-domain antibodies (sdAbs) targeting both protein- and peptide antigens. We find clear patterns in the number antibody-antigen contacts and amino acid frequencies in the paratope. The direct comparison of sdAbs and conventional antibodies helps elucidate the mechanisms employed by sdAbs to compensate for their smaller size and the fact that they harbor only half the number of complementarity-determining regions compared to conventional antibodies. Furthermore, we pinpoint antibody interface hotspot residues that are often found at the binding interface and the amino acid frequencies at these positions. These findings have direct potential applications in antibody engineering and the design of improved antibody libraries.

Aluminum nitride (AlN) is a material of growing interest for power electronics, fabrication of sensors, micro-electromechanical systems, and piezoelectric generators. For the latter, the formation of nanowire arrays or nanostructured films is one of the emerging research directions. In the current work, nanostructured AlN films manufactured with normal and glancing angle magnetron sputter depositions have been investigated with scanning and transmission electron microscopy, X-ray diffraction, atomic force microscopy, and optical spectroscopy. Growth of the nanostructures was realized utilizing metal seed particles (Ag, Au, and Al), allowing the control of the nucleation and following growth of AlN. It was demonstrated how variations of seed particle material and size can be used to tune the parameters of nanostructures and morphology of the AlN films. Using normal angle deposition allowed the growth of bud-shaped structures, which consisted of pillars/lamellae with wurtzite-like crystalline structures. Deposition at a glancing angle of 85° led to a film of individual nanostructures located near each other and tilted at an angle of 33 degree; relative to the surface normal. Such films maintained a high degree of wurtzite-like crystallinity but had a more open structure and higher roughness than the nanostructured films grown at normal incidence deposition. The developed production strategies and recipes for controlling parameters of nanostructured films pave the way for the formation of matrices to be used in piezoelectric applications., Novo Nordisk Foundation

Introduction: Diabetes is associated with dysregulated immune function and impaired cytokine release, while transient acute hyperglycaemia has been shown to enhance inflammatory cytokine release in preclinical studies. Although diabetes and acute hyperglycaemia are common among patients with community-acquired pneumonia (CAP), the impact of chronic, acute, and acute-on-chronic hyperglycaemia on the host response within this population remains poorly understood. This study investigated whether chronic, acute, and acute-on- chronic hyperglycaemia are associated with distinct mediators of inflammatory, endothelial, and angiogenic host response pathways in patients with CAP. Methods: In a cross-sectional study of 555 patients with CAP, HbA1c, admission plasma (p)-glucose, and the glycaemic gap (admission p-glucose minus HbA1c- derived average p-glucose) were employed as measures of chronic, acute, and acute-on-chronic hyperglycaemia, respectively. Linear regression was used to model the associations between the hyperglycaemia measures and 47 proteins involved in inflammation, endothelial activation, and angiogenesis measured at admission. The models were adjusted for age, sex, CAP severity, pathogen, immunosuppression, comorbidity, and body mass index. Adjustments for multiple testing were performed with a false discovery rate threshold of less than 0.05. Results: The analyses showed that HbA1c levels were positively associated with IL-8, IL-15, IL-17A/F, IL-1RA, sFlt-1, and VEGF-C. Admission plasma glucose was also positively associated with these proteins and GM-CSF. The glycaemic gap was positively associated with IL-8, IL-15, IL-17A/F, IL-2, and VEGF-C. Conclusion: In conclusion, chronic, acute, and acute-on-chronic hyperglycaemia were positively associated with similar host response mediators. Furthermore, acute and acute-on-chronic hyperglycaemia had unique associations with the inflammatory pathways involving GM-CSF and IL-2, respective, Introduction: Diabetes is associated with dysregulated immune function and impaired cytokine release, while transient acute hyperglycaemia has been shown to enhance inflammatory cytokine release in preclinical studies. Although diabetes and acute hyperglycaemia are common among patients with community-acquired pneumonia (CAP), the impact of chronic, acute, and acute-on-chronic hyperglycaemia on the host response within this population remains poorly understood. This study investigated whether chronic, acute, and acute-on- chronic hyperglycaemia are associated with distinct mediators of inflammatory, endothelial, and angiogenic host response pathways in patients with CAP. Methods: In a cross-sectional study of 555 patients with CAP, HbA1c, admission plasma (p)-glucose, and the glycaemic gap (admission p-glucose minus HbA1c- derived average p-glucose) were employed as measures of chronic, acute, and acute-on-chronic hyperglycaemia, respectively. Linear regression was used to model the associations between the hyperglycaemia measures and 47 proteins involved in inflammation, endothelial activation, and angiogenesis measured at admission. The models were adjusted for age, sex, CAP severity, pathogen, immunosuppression, comorbidity, and body mass index. Adjustments for multiple testing were performed with a false discovery rate threshold of less than 0.05. Results: The analyses showed that HbA1c levels were positively associated with IL-8, IL-15, IL-17A/F, IL-1RA, sFlt-1, and VEGF-C. Admission plasma glucose was also positively associated with these proteins and GM-CSF. The glycaemic gap was positively associated with IL-8, IL-15, IL-17A/F, IL-2, and VEGF-C. Conclusion: In conclusion, chronic, acute, and acute-on-chronic hyperglycaemia were positively associated with similar host response mediators. Furthermore, acute and acute-on-chronic hyperglycaemia had unique associations with the inflammatory pathways involving GM-CSF and IL-2, respectively.

Background Worldwide, up to 20 % of hospitalised patients have diabetes mellitus. In-hospital dysglycaemia increases patient mortality, morbidity, and length of hospital stay. Improved in-hospital diabetes management strategies are needed. The DIATEC trial investigates the effects of an in-hospital diabetes team and operational insulin titration algorithms based on either continuous glucose monitoring (CGM) data or standard point-of-care (POC) glucose testing. Methods This is a two-armed, two-site, prospective randomised open-label blinded endpoint (PROBE) trial. We recruit non-critically ill hospitalised general medical and orthopaedic patients with type 2 diabetes treated with basal, prandial, and correctional insulin (N = 166). In both arms, patients are monitored by POC glucose testing and diabetes management is done by ward nurses guided by in-hospital diabetes teams. In one of the arms, patients are monitored in addition to POC glucose testing by telemetric CGM viewed by the in-hospital diabetes teams only. The in-hospital diabetes teams have operational algorithms to titrate insulin in both arms. Outcomes are in-hospital glycaemic and clinical outcomes. Discussion The DIATEC trial will show the glycaemic and clinical effects of in-hospital CGM handled by in-hospital diabetes teams with access to operational insulin titration algorithms in non-critically ill patients with type 2 diabetes. The DIATEC trial seeks to identify which hospitalised patients will benefit from CGM and in-hospital diabetes teams compared to POC glucose testing. This is essential information to optimise the use of healthcare resources before broadly implementing in-hospital CGM and diabetes teams., Background: Worldwide, up to 20 % of hospitalised patients have diabetes mellitus. In-hospital dysglycaemia increases patient mortality, morbidity, and length of hospital stay. Improved in-hospital diabetes management strategies are needed. The DIATEC trial investigates the effects of an in-hospital diabetes team and operational insulin titration algorithms based on either continuous glucose monitoring (CGM) data or standard point-of-care (POC) glucose testing. Methods: This is a two-armed, two-site, prospective randomised open-label blinded endpoint (PROBE) trial. We recruit non-critically ill hospitalised general medical and orthopaedic patients with type 2 diabetes treated with basal, prandial, and correctional insulin (N = 166). In both arms, patients are monitored by POC glucose testing and diabetes management is done by ward nurses guided by in-hospital diabetes teams. In one of the arms, patients are monitored in addition to POC glucose testing by telemetric CGM viewed by the in-hospital diabetes teams only. The in-hospital diabetes teams have operational algorithms to titrate insulin in both arms. Outcomes are in-hospital glycaemic and clinical outcomes. Discussion: The DIATEC trial will show the glycaemic and clinical effects of in-hospital CGM handled by in-hospital diabetes teams with access to operational insulin titration algorithms in non-critically ill patients with type 2 diabetes. The DIATEC trial seeks to identify which hospitalised patients will benefit from CGM and in-hospital diabetes teams compared to POC glucose testing. This is essential information to optimise the use of healthcare resources before broadly implementing in-hospital CGM and diabetes teams. Trial registration: Prospectively registered at ClinicalTrials.gov with identification number NCT05803473 on March 27th 2023.

Background: Underweight and obesity are associated with adverse health outcomes throughout the life course. We estimated the individual and combined prevalence of underweight or thinness and obesity, and their changes, from 1990 to 2022 for adults and school-aged children and adolescents in 200 countries and territories. Methods: We used data from 3663 population-based studies with 222 million participants that measured height and weight in representative samples of the general population. We used a Bayesian hierarchical model to estimate trends in the prevalence of different BMI categories, separately for adults (age ≥20 years) and school-aged children and adolescents (age 5–19 years), from 1990 to 2022 for 200 countries and territories. For adults, we report the individual and combined prevalence of underweight (BMI <18·5 kg/m2) and obesity (BMI ≥30 kg/m2). For school-aged children and adolescents, we report thinness (BMI <2 SD below the median of the WHO growth reference) and obesity (BMI >2 SD above the median). Findings: From 1990 to 2022, the combined prevalence of underweight and obesity in adults decreased in 11 countries (6%) for women and 17 (9%) for men with a posterior probability of at least 0·80 that the observed changes were true decreases. The combined prevalence increased in 162 countries (81%) for women and 140 countries (70%) for men with a posterior probability of at least 0·80. In 2022, the combined prevalence of underweight and obesity was highest in island nations in the Caribbean and Polynesia and Micronesia, and countries in the Middle East and north Africa. Obesity prevalence was higher than underweight with posterior probability of at least 0·80 in 177 countries (89%) for women and 145 (73%) for men in 2022, whereas the converse was true in 16 countries (8%) for women, and 39 (20%) for men. From 1990 to 2022, the combined prevalence of thinness and obesity decreased among girls in five countries (3%) and a

Objectives Physical activity is associated with improved health in people with type 1 diabetes. However, physical activity level may be associated with socioeconomic status. The primary aim was to investigate the association between education level and physical activity level amongst people with type 1 diabetes. Methods In this cross-sectional study, data on physical activity level (high or low) was measured using the Saltin–Grimby Physical Activity Level Scale, and education level (low, medium, or high) was self-reported. Results Respondents were recruited from outpatient clinics (Steno Diabetes Centre Aarhus (DK), Nordsjællands Hospital (DK) or Sheffield Diabetes and Endocrine Centre (UK)), by healthcare personnel from September 2019 to July 2021. 324 people with type 1 diabetes were included (54% male, median age 50 years (IQR 30-60)). Education level was low in 10%, medium in 33%, and high in 57%. A logistic regression analysis, adjusted for age, sex, cohabitation status and nationality, found that a medium vs. high education level was associated with lower odds of a high physical activity level OR 0.55 [95% CI 0.32;0.94], p=0.029, while no association was found for low vs. high education level with high physical activity level (OR 0.56 [0.25; 1.29], p=0.173). Conclusions Medium education level compared with a high education level was associated with a lower level of physical activity in people with type 1 diabetes. Healthcare professionals are advised to be attentive of physical activity levels amongst people type 1 diabetes. The study was approved by the Danish Data Protection Agency: P-2016-48.s, Objectives: Physical activity is associated with improved health in people with type 1 diabetes. However, physical activity level may be associated with socioeconomic status. The primary aim was to investigate the association between education level and physical activity level amongst people with type 1 diabetes. Methods: In this cross-sectional study, data on physical activity level (high or low) was measured using the Saltin–Grimby Physical Activity Level Scale, and education level (low, medium, or high) was self-reported. Results: Respondents were recruited from outpatient clinics (Steno Diabetes Centre Aarhus (DK), Nordsjællands Hospital (DK) or Sheffield Diabetes and Endocrine Centre (UK)), by healthcare personnel from September 2019 to July 2021. 324 people with type 1 diabetes were included (54% male, median age 50 years (IQR 30-60)). Education level was low in 10%, medium in 33%, and high in 57%. A logistic regression analysis, adjusted for age, sex, cohabitation status and nationality, found that a medium vs. high education level was associated with lower odds of a high physical activity level OR 0.55 [95% CI 0.32;0.94], p=0.029, while no association was found for low vs. high education level with high physical activity level (OR 0.56 [0.25; 1.29], p=0.173). Conclusions: Medium education level compared with a high education level was associated with a lower level of physical activity in people with type 1 diabetes. Healthcare professionals are advised to be attentive of physical activity levels amongst people type 1 diabetes. The study was approved by the Danish Data Protection Agency: P-2016-48.s Sponsor: Peter Lommer Kristensen.

Aim: The sympathetic nervous and hormonal counterregulatory responses to hypoglycaemia differ between people with type 1 and type 2 diabetes and may change along the course of diabetes, but have not been directly compared. We aimed to compare counterregulatory hormone and symptom responses to hypoglycaemia between people with type 1 diabetes, insulin-treated type 2 diabetes and controls without diabetes, using a standardised hyperinsulinaemic-hypoglycaemic clamp. Materials: We included 47 people with type 1 diabetes, 15 with insulin-treated type 2 diabetes, and 32 controls without diabetes. Controls were matched according to age and sex to the people with type 1 diabetes or with type 2 diabetes. All participants underwent a hyperinsulinaemic–euglycaemic-(5.2 ± 0.4 mmol/L)-hypoglycaemic-(2.8 ± 0.13 mmol/L)-clamp. Results: The glucagon response was lower in people with type 1 diabetes (9.4 ± 0.8 pmol/L, 8.0 [7.0–10.0]) compared to type 2 diabetes (23.7 ± 3.7 pmol/L, 18.0 [12.0–28.0], p < 0.001) and controls (30.6 ± 4.7, 25.5 [17.8–35.8] pmol/L, p < 0.001). The adrenaline response was lower in type 1 diabetes (1.7 ± 0.2, 1.6 [1.3–5.2] nmol/L) compared to type 2 diabetes (3.4 ± 0.7, 2.6 [1.3–5.2] nmol/L, p = 0.001) and controls (2.7 ± 0.4, 2.8 [1.4–3.9] nmol/L, p = 0.012). Growth hormone was lower in people with type 2 diabetes than in type 1 diabetes, at baseline (3.4 ± 1.6 vs 7.7 ± 1.3 mU/L, p = 0.042) and during hypoglycaemia (24.7 ± 7.1 vs 62.4 ± 5.8 mU/L, p = 0.001). People with 1 diabetes had lower overall symptom responses than people with type 2 diabetes (45.3 ± 2.7 vs 58.7 ± 6.4, p = 0.018), driven by a lower neuroglycopenic score (27.4 ± 1.8 vs 36.7 ± 4.2, p = 0.012). Conclusion: Acute counterregulatory hormone and symptom responses to experimental hypoglycaemia are lower in people with type 1 diabetes than in those with long-standing insulin-treated type 2 diabetes and controls.

C-reactive protein (CRP) is commonly used to guide community-acquired pneumonia (CAP) treatment. A positive association between admission glucose and CRP levels has been observed in patients with CAP. The associations between prediabetes, unknown diabetes, acute-on-chronic hyperglycaemia, and CRP levels, and between admission CRP levels and insulin resistance (IR) in CAP, remain unexplored. This study investigated the associations firstly between chronic, acute, and acute-on-chronic hyperglycaemia and CRP levels, and secondly between admission CRP levels and IR in CAP. In a prospective cohort study of adults with CAP, the associations between chronic, acute, and acute-on-chronic hyperglycaemia (admission glucose minus HbA1c-derived average glucose) and CRP levels until admission day 3 were modelled with repeated-measures linear mixed models. IR was estimated with the homeostasis model assessment of IR (HOMA-IR). The association between admission CRP levels and HOMA-IR was modelled with linear regression. In 540 patients, no association between chronic, acute, or acute-on-chronic hyperglycaemia and CRP levels was found. In 266 patients, every 50 mg/L increase in admission CRP was associated with a 7% (95% CI 1–14%) higher HOMA-IR. In conclusion, our findings imply that hyperglycaemia does not influence CRP levels in patients with CAP, although admission CRP levels were positively associated with IR. Keywords: community-acquired pneumonia; c-reactive protein; diabetes mellitus; chronic hyperglycaemia; acute hyperglycaemia; acute-on-chronic hyperglycaemia; insulin resistance, C-reactive protein (CRP) is commonly used to guide community-acquired pneumonia (CAP) treatment. A positive association between admission glucose and CRP levels has been observed in patients with CAP. The associations between prediabetes, unknown diabetes, acute-on-chronic hyperglycaemia, and CRP levels, and between admission CRP levels and insulin resistance (IR) in CAP, remain unexplored. This study investigated the associations firstly between chronic, acute, and acute-on-chronic hyperglycaemia and CRP levels, and secondly between admission CRP levels and IR in CAP. In a prospective cohort study of adults with CAP, the associations between chronic, acute, and acute-on-chronic hyperglycaemia (admission glucose minus HbA1c-derived average glucose) and CRP levels until admission day 3 were modelled with repeated-measures linear mixed models. IR was estimated with the homeostasis model assessment of IR (HOMA-IR). The association between admission CRP levels and HOMA-IR was modelled with linear regression. In 540 patients, no association between chronic, acute, or acute-on-chronic hyperglycaemia and CRP levels was found. In 266 patients, every 50 mg/L increase in admission CRP was associated with a 7% (95% CI 1–14%) higher HOMA-IR. In conclusion, our findings imply that hyperglycaemia does not influence CRP levels in patients with CAP, although admission CRP levels were positively associated with IR.

Although DNA‐based cancer vaccines have been successfully tested in mouse models, a major drawback of cancer vaccination still remains, namely that tumour antigens are weak and fail to generate a vigorous immune response in tumour‐bearing patients. Genetic technology offers strategies for promoting immune pathways by adding immune‐activating genes to the tumour antigen sequence. In this work, we converted a model non‐immunogenic antigen into a vaccine by fusing it to domain I of the filamentous bacteriophage coat protein III gene. Vaccination with a DNA construct encoding the domain I fusion generated antigen‐specific T helper 1‐type cellular immune responses. These results demonstrate that the incorporation of protein III into a DNA vaccine formulation can modulate the gene‐mediated immune response and may thus provide a strategy for improving its therapeutic effect., Depto. de Bioquímica y Biología Molecular, Fac. de Farmacia, TRUE, pub

In issues of war and peace, women have traditionally been excluded from the conversation and their gender-specific experiences have been excluded in the process. In 2000, Resolution 1325 contributed to the discussion of women's equal participation, inclusion and protection and towards a positive trend of higher rates of women's inclusion. However, in recent years these numbers have started to drop again and putting women's issues on the agenda has remained challenging. Even though more and more research concerns itself with women's representation, a detailed understanding of the types of inclusion and the effects hereof on durable peace, remain limited. As such, this thesis explores the relation between Ellerby's (2013) four properties of inclusion under (en)gendered security: 1) representation, 2) incorporation, 3) protection, and 4) recognition and the levels of durable peace experienced by women after the signing of the peace agreements. Theoretically, informed by a univerlistic value basis, an expansion of liberal feminist ideals is suggested to be able to consider and focus on women's rights and issues. Through purposive sampling and a small sample comparative case study, the Guatemalan and Colombian peace agreements were selected as the cases for the base of the analysis, and supported by document analysis. The analysis is based on a combination of primary and secondary data, in the form of the peace agreements, official reports, books, journal articles, and more. Analytically, this thesis aims to uncover how women were included in the peace agreements and women's current day-to-day lives in order to get a sense of the level of durable peace and what can be learned from the similarities and differences between the selected cases. Based on the findings, this thesis concludes that the Colombian peace agreement can be classified as a more durable peace, as a result of the high levels of inclusion of women and the specific laws, measures, and implementations prese

Governments increasingly draw inspiration from industrial policy in the design of new climate policies. The Inflation Reduction Act of 2022 (IRA) exemplifies the protectionist logic of industrial policy on the largest scale yet. The provisions of the legislation reflect a clear goal to revitalize American manufacturing, by supporting clean-tech manufacturers and sustainable energy suppliers through subsidies with American content requirements. This has important implications for future international climate cooperation, as it distorts competition in international trade to the benefit of the subsidizing state. This is further complicated if a trading partner seeks to impose costs on national manufacturers, e.g., by carbon taxes. We argue that these aspects are important to study to understand the future possibilities of selective climate cooperation, where like-minded states may form Climate Clubs. Yet, the literature composing the field of Global Climate Governance lacks descriptions of how industrial policy may be incorporated in climate policies. The field largely views climate governance through a set of ‘modes’ that address questions of climate diplomacy behind international agreements and economic cooperation through carbon pricing. We argue that it is important to understand the effects of domestic policy, when seeking to understand developments on the global level. To bridge the knowledge gap concerning industrial policy in climate governance we construct a new mode; The Green Industrial Policy mode of global climate governance. We apply this to analyze the policy contents of the IRA and its official justification, whereby we find that the policy is highly designed to achieve economic growth and energy independence through supporting strategic industries, initially disregarding the consequences of trading partners in an aim to re-shore American manufacturing jobs exported to China. We find that Green Industrial Policy designs may fragment climate cooperation