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On the day he went missing, she went to the podiatrist. She took the bus. She visited the podiatrist regularly, ever since Rob ran over her foot backing the car [...]

Abstract Precise epitope determination of therapeutic antibodies is of great value as it allows for further comprehension of mechanism of action, therapeutic responsiveness prediction, avoidance of unwanted cross reactivity, and vaccine design. The golden standard for discontinuous epitope determination is the laborious X-ray crystallography method. Here, we present a combinatorial method for rapid mapping of discontinuous epitopes by mammalian antigen display, eliminating the need for protein expression and purification. The method is facilitated by automated workflows and tailored software for antigen analysis and oligonucleotide design. These oligos are used in automated mutagenesis to generate an antigen receptor library displayed on mammalian cells for direct binding analysis by flow cytometry. Through automated analysis of 33930 primers an optimized single condition cloning reaction was defined allowing for mutation of all surface-exposed residues of the receptor binding domain of SARS-CoV-2. All variants were functionally expressed, and two reference binders validated the method. Furthermore, epitopes of three novel therapeutic antibodies were successfully determined followed by evaluation of binding also towards SARS-CoV-2 Omicron BA.2. We find the method to be highly relevant for rapid construction of antigen libraries and determination of antibody epitopes, especially for the development of therapeutic interventions against novel pathogens.

Abstract Streptococcus pyogenes can cause invasive disease with high mortality despite adequate antibiotic treatments. To address this unmet need, we have previously generated an opsonic IgG1 monoclonal antibody, Ab25, targeting the bacterial M protein. Here, we engineer the IgG2-4 subclasses of Ab25. Despite having reduced binding, the IgG3 version promotes stronger phagocytosis of bacteria. Using atomic simulations, we show that IgG3’s Fc tail has extensive movement in 3D space due to its extended hinge region, possibly facilitating interactions with immune cells. We replaced the hinge of IgG1 with four different IgG3-hinge segment subclasses, IgGhxx. Hinge-engineering does not diminish binding as with IgG3 but enhances opsonic function, where a 47 amino acid hinge is comparable to IgG3 in function. IgGh47 shows improved protection against S. pyogenes in a systemic infection mouse model, suggesting that IgGh47 has promise as a preclinical therapeutic candidate. Importantly, the enhanced opsonic function of IgGh47 is generalizable to diverse S. pyogenes strains from clinical isolates. We generated IgGh47 versions of anti-SARS-CoV-2 mAbs to broaden the biological applicability, and these also exhibit strongly enhanced opsonic function compared to the IgG1 subclass. The improved function of the IgGh47 subclass in two distant biological systems provides new insights into antibody function.

IntroductionFish β-parvalbumins are common targets of allergy-causing immunity. The nature of antibody responses to such allergens determines the biological outcome following exposure to fish. Specific epitopes on these allergens recognised by antibodies are incompletely characterised.MethodsHigh-content peptide microarrays offer a solution to the identification of linear epitopes recognised by antibodies. We characterized IgG and IgG4 recognition of linear epitopes of fish β-parvalbumins defined in the WHO/IUIS allergen database as such responses hold the potential to counter an allergic reaction to these allergens. Peripheral blood samples, collected over three years, of 15 atopic but not fish-allergic subjects were investigated using a microarray platform that carried every possible 16-mer peptide of known isoforms and isoallergens of these and other allergens.ResultsInterindividual differences in epitope recognition patterns were observed. In contrast, reactivity patterns in a given individual were by comparison more stable during the 3 years-course of the study. Nevertheless, evidence of the induction of novel specificities over time was identified across multiple regions of the allergens. Particularly reactive epitopes were identified in the D helix of Cyp c 1 and in the C-terminus of Gad c 1 and Gad m 1.02. Residues important for the recognition of certain linear epitopes were identified. Patterns of differential recognition of isoallergens were observed in some subjects.ConclusionsAltogether, comprehensive analysis of antibody recognition of linear epitopes of multiple allergens enables characterisation of the nature of the antibody responses targeting this important set of food allergens.

Abstract We report the application of ancestral sequence reconstruction on coronavirus spike protein, resulting in stable and highly soluble ancestral scaffold antigens (AnSAs). The AnSAs interact with plasma of patients recovered from COVID-19 but do not bind to the human angiotensin-converting enzyme 2 (ACE2) receptor. Cryo-EM analysis of the AnSAs yield high resolution structures (2.6–2.8 Å) indicating a closed pre-fusion conformation in which all three receptor-binding domains (RBDs) are facing downwards. The structures reveal an intricate hydrogen-bonding network mediated by well-resolved loops, both within and across monomers, tethering the N-terminal domain and RBD together. We show that AnSA-5 can induce and boost a broad-spectrum immune response against the wild-type RBD as well as circulating variants of concern in an immune organoid model derived from tonsils. Finally, we highlight how AnSAs are potent scaffolds by replacing the ancestral RBD with the wild-type sequence, which restores ACE2 binding and increases the interaction with convalescent plasma.

This study takes its departure from ongoing debate about teachers' (collective) "continuing professional development" (CPD). The overall aim is to highlight active teachers' perceptions on developing a self-understanding of their complex role in daily practice by using digital tools. The following research questions guided the study: How can teachers make schools more relevant and engaging? How can students' achievement increase? In what way can teachers provide high-quality education for all students? Three perspectives will be guiding the study: Information and Communication Technology (ICT), learning, and special education. The participants in the study are 21 staff members in preschool, primary school, and a recreation center with whom we conducted structured individual interviews and focus group discussions. Field notes were also taken during the interview and discussion sessions. The findings reveal the importance of the teachers' deeper understanding of students' creativity, critical thinking, communication, and collaboration. Furthermore, the most powerful thing teachers can do is to design engaging, meaningful, and authentic work and technology-enhanced learning experiences. In order to improve learning in a digital world, the teachers must be engaged and supported by professional learning opportunities to continually improve and strengthen their digital competencies and teaching practices. The theoretical standpoint is the norm model as an analytical tool to understand the teachers' perceptions. The concept of "norm" is a collective term for the factors and structures that are regarded as a normal balance between the aspects of value/will, system conditions/possibilities, and cognition/knowledge.

Abstract This study aimed to establish the cost-effectiveness of home phototherapy versus hospital phototherapy treating hyperbilirubinemia in neonates more than 36 weeks. Based on clinical results from a randomised controlled trial showing that home phototherapy for hyperbilirubinemia in term neonates is as effective as hospital phototherapy, we performed a cost-minimisation analysis to identify the most cost-effective alternative. We included costs for health care resource use as well as costs for transportation in connection with re-visits. The cost per patient was €337 for home phototherapy compared with €1156 for the hospital alternative indicating average cost savings of €819 (95% confidence interval €613–1025) or 71% per patient. Transportation and outpatient costs were higher in the home treatment group and hospital care costs were higher in the hospital group. Sensitivity analysis shows that results are robust also when allowing for uncertainty. Home phototherapy for neonates more than 36 weeks costs less than in-hospital phototherapy while being equally effective, meaning that home phototherapy is a cost-effective alternative to hospital treatment for infants with neonatal hyperbilirubinemia. Trial registration NCT03536078 . Date of registration: 24/05/2018.

IntroductionAnalysis of an individual’s immunoglobulin (IG) gene repertoire requires the use of high-quality germline gene reference sets. When sets only contain alleles supported by strong evidence, AIRR sequencing (AIRR-seq) data analysis is more accurate and studies of the evolution of IG genes, their allelic variants and the expressed immune repertoire is therefore facilitated.MethodsThe Adaptive Immune Receptor Repertoire Community (AIRR-C) IG Reference Sets have been developed by including only human IG heavy and light chain alleles that have been confirmed by evidence from multiple high-quality sources. To further improve AIRR-seq analysis, some alleles have been extended to deal with short 3’ or 5’ truncations that can lead them to be overlooked by alignment utilities. To avoid other challenges for analysis programs, exact paralogs (e.g. IGHV1-69*01 and IGHV1-69D*01) are only represented once in each set, though alternative sequence names are noted in accompanying metadata.Results and discussionThe Reference Sets include less than half the previously recognised IG alleles (e.g. just 198 IGHV sequences), and also include a number of novel alleles: 8 IGHV alleles, 2 IGKV alleles and 5 IGLV alleles. Despite their smaller sizes, erroneous calls were eliminated, and excellent coverage was achieved when a set of repertoires comprising over 4 million V(D)J rearrangements from 99 individuals were analyzed using the Sets. The version-tracked AIRR-C IG Reference Sets are freely available at the OGRDB website (https://ogrdb.airr-community.org/germline_sets/Human) and will be regularly updated to include newly observed and previously reported sequences that can be confirmed by new high-quality data.

Background:. Lymphedema leads to adipose tissue deposition that cannot be removed using conservative methods. Previous studies have shown a complete reduction in excess volume in limbs with lymphedema when treated with liposuction and controlled compression therapy (CCT). We present the long-term outcomes of all patients treated with liposuction and CCT for lower extremity lymphedema (LEL) who were followed up for 5 years. Methods:. Sixty-seven LEL patients underwent liposuction and CCT. Thirty-six patients had primary lymphedema and 31 patients had secondary lymphedema. The outcomes included excess leg volume over a follow-up period of 5 years. Any association between patient characteristics and treatment outcomes was analyzed. Results:. The preoperative excess volume prior was 3515 mL [interquartile range (IQR): 2225–5455 mL], and the volume ratio to the unaffected leg was 1.35 (IQR: 1.25–1.53). One year after treatment, the excess volume decreased by 101% (IQR: 84–116). The decrease in excess volume continued during the 5-year follow-up, and at the end of the study, the excess volume had decreased by 115% (IQR: 98–124). No major complications were noted. Conclusions:. Liposuction and CCT are safe and effective procedures for removing excess adipose tissue and normalizing the leg volume in patients with late-stage LEL. When no satisfactory results are obtained with conservative methods, such as complex decongestive therapy, and there is no or minimal pitting on limb examination, excess adipose tissue is present, and liposuction can be considered.

Summary: Susceptibility to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections is highly variable and could be mediated by a cross-protective pre-immunity. We identified 14 cross-reactive peptides between SARS-CoV-2 and influenza A H1N1, H3N2, and human herpesvirus (HHV)-6A/B with potential relevance. The H1N1 peptide NGVEGF was identical to a peptide in the most critical receptor binding motif in SARS-CoV-2 spike protein that interacts with the angiotensin converting enzyme 2 receptor. About 62%–73% of COVID-19-negative blood donors in Stockholm had antibodies to this peptide in the early pre-vaccination phase of the pandemic. Seasonal flu vaccination enhanced neutralizing capacity to SARS-CoV-2 and T cell immunity to this peptide. Mathematical modeling taking the estimated pre-immunity levels to flu into account could fully predict pre-Omicron SARS-CoV-2 outbreaks in Stockholm and India. This cross-immunity provides mechanistic explanations to the epidemiological observation that influenza vaccination protected people against early SARS-CoV-2 infections and implies that flu-mediated cross-protective immunity significantly dampened the first SARS-CoV-2 outbreaks.

Metal assisted chemical etching is a promising method for fabricating high aspect ratio micro- and nanostructures in silicon. Previous results have suggested that P-type and N-type silicon etches with different degrees of anisotropy, questioning the use of P-type silicon for nanostructures. In this study, we compare processing X-ray zone plate nanostructures in N and P-type silicon through metal assisted chemical etching with a gold catalyst. Fabricated zone plates were cleaved and studied with a focus on resulting verticality, depth and porosity. Results show that for high aspect ratio nanostructures, both N and P-type silicon prove to be viable alternatives exhibiting different etch rates, but similarities regarding porosity and etch direction.

Controlling and understanding charge state and metal coordination in carbon nanomaterials is crucial to harnessing their unique properties. Here we describe the synthesis of the well‐defined fulleride complex [{(Mesnacnac)Mg}6C70], 2, (Mesnacnac)=HC(MeCNMes)2, Mes=2,4,6‐Me3C6H2, from the reaction of the β‐diketiminate magnesium(I) complex [{(Mesnacnac)Mg}2] with C70 in aromatic solvents. The molecular structure of complex 2 was determined, providing the first high‐quality structural study of a complex with the C706− ion. In combination with solution state NMR spectroscopic and DFT computational studies, the changes in geometry and charge distribution in the various atom and bond types of the fulleride unit were investigated. Additionally, the influence of the (Mesnacnac)Mg+ cations on the global and local fulleride coordination environment was examined. [ABSTRACT FROM AUTHOR]

Australia's bushfoods, botanicals and traditional medicines sector are a growing industry based on thousands of years of Indigenous Knowledge (IK). National and international growth in this industry does not provide satisfactory or equitable sharing of benefits, preventing opportunities and funding for Aboriginal and Torres Strait Islander peoples. Without prior informed consent (PIC) and access and benefit sharing (ABS) arrangements, alongside specific point of difference certification and labelling schemes, current industry inequities will continue. International protocols such as the Nagoya Protocol require ABS arrangements, and we argue whether a certification trademark (CTM) scheme could also support ABS in Australia. ABS lays the groundwork to ethically share IK to mitigate harms and provide benefits to Aboriginal and Torres Strait Islander peoples. Establishing coherent CTM and labelling schemes will complement and could work towards addressing the gaps and limitations in Australian ABS laws. CTM and labelling systems, as well as strategies and statements used by established alliances and organisations, provide insight into what mechanisms current ABS laws allow for. We use these examples to recommend the next steps. [ABSTRACT FROM AUTHOR]

The fractionation factor in the magnesium-isotope fractionation between aqueous solutions of magnesium and brucite changes sign with increasing temperature, as uncovered by recent experiments. To understand this behavior, the Reduced Partition Function Ratios and isotopic fractionation factors (Δ26/24Mgbrucite-Mg(aq)) are calculated using molecular models of aqueous [Mg(OH2)6]2+ and the mineral brucite at increasing levels of density functional theory. The calculations were carried out on the [Mg(OH2)6]2+·12H2O cluster, along with different Pauling-bond-strength-conserving models of the mineral lattice of brucite. Three conclusions were reached: (i) all levels of theory overestimate 〈Mg–O〉 bond distances in the aqua ion complex relative to Tutton's salts; (ii) the calculations predict that brucite at 298.15 K is always enriched in the heavy isotope, in contrast with experimental observations; (iii) the temperature dependencies of Wimpenny et al. (2014) and Li et al. (2014) could only be achieved by fixing the 〈Mg–O〉 bond distances in the [Mg(OH2)6]2+·12H2O cluster to values close to those observed in crystals that trap the hydrated ion.

The fractionation factor in the magnesium-isotope fractionation between aqueous solutions of magnesium and brucite changes sign with increasing temperature, as uncovered by recent experiments. To understand this behavior, the Reduced Partition Function Ratios and isotopic fractionation factors (Δ26/24Mgbrucite-Mg(aq)) are calculated using molecular models of aqueous [Mg(OH2)6]2+ and the mineral brucite at increasing levels of density functional theory. The calculations were carried out on the [Mg(OH2)6]2+·12H2O cluster, along with different Pauling-bond-strength-conserving models of the mineral lattice of brucite. Three conclusions were reached: (i) all levels of theory overestimate 〈Mg–O〉 bond distances in the aqua ion complex relative to Tutton's salts; (ii) the calculations predict that brucite at 298.15 K is always enriched in the heavy isotope, in contrast with experimental observations; (iii) the temperature dependencies of Wimpenny et al. (2014) and Li et al. (2014) could only be achieved by fixing the 〈Mg–O〉 bond distances in the [Mg(OH2)6]2+·12H2O cluster to values close to those observed in crystals that trap the hydrated ion.