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Delineation of structural variants (SVs) at sequence resolution in highly repetitive genomic regions has long been intractable. The sequence properties, origins, and functional effects of classes of genomic rearrangements such as ring chromosomes and Robertsonian translocations thus remain unknown. To resolve these complex structures, we leveraged several recent milestones in the field, including (1) the emergence of long-read sequencing, (2) the gapless telomere-to-telomere (T2T) assembly, and (3) a tool (BigClipper) to discover chromosomal rearrangements from long reads. We applied these technologies across 13 cases with ring chromosomes, Robertsonian translocations, and complex SVs that were unresolved by short reads, followed by validation using optical genome mapping (OGM). Our analyses resolved 10 of 13 cases, including a Robertsonian translocation and all ring chromosomes. Multiple breakpoints were localized to genomic regions previously recalcitrant to sequencing such as acrocentric p-arms, ribosomal DNA arrays, and telomeric repeats, and involved complex structures such as a deletion-inversion and interchromosomal dispersed duplications. We further performed methylation profiling from long-read data to discover phased differential methylation in a gene promoter proximal to a ring fusion, suggesting a long-range position effect (LRPE) with heterochromatin spreading. Breakpoint sequences suggested mechanisms of SV formation such as microhomology-mediated and non-homologous end-joining, as well as non-allelic homologous recombination. These methods provide some of the first glimpses into the sequence resolution of Robertsonian translocations and illuminate the structural diversity of ring chromosomes and complex chromosomal rearrangements with implications for genome biology, prediction of LRPEs from integrated multi-omics technologies, and molecular diagnostics in rare disease cases., Competing Interests: Declaration of interests The Talkowski laboratory receives reagents and/or research support from Illumina Inc and Microsoft Inc. Bionano Genomics provided data and analysis support for Optical Genome Mapping. J.H. and A.R.H. are employees of Bionano Genomics, Inc., (Copyright © 2024 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Plant cell walls are complex, multifunctional structures, built up of polysaccharides and proteins. The configuration and abundance of cell wall constituents determine cellular elongation and plant growth. The emphasis of this review is on rice, a staple crop with economic importance, serving as model for grasses/cereals. Recent advancements have contributed to a better understanding of the grass/cereal cell wall. This review brings together current knowledge of the organization and metabolism of the rice cell wall, and addresses gaps in the information regarding the cell wall and enzymes involved. Several cell wall fractions, including cellulose, mixed-linkage glucans, and glucuronoarabinoxylans, are well understood in rice and other grasses/grains. Conversely, there are still open questions and missing links in relation to xyloglucans, glucomannans, pectin, lignin, and arabinogalactan proteins. There is still a large and untapped potential to identify carbohydrate-active enzymes (CAZymes), to characterize their activity, and to elucidate their involvement in the metabolism of the mentioned cell wall fractions. This review highlights the involvement of carbohydrate-active enzymes in rice cell wall metabolism, providing an update of current understanding with the aim of demarcating research areas with potential for further investigations., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Aims: Decision-makers still predominantly focus on the perspective of non-patient stakeholders, which may deviate from the unique perspective of heart failure (HF) patients. To enhance patient-centred decision-making, there is a need for more patient-based evidence derived directly from the patients themselves. Hence, this study aimed to understand (i) HF patients' unmet medical needs and preferred treatment outcomes; (ii) patients' risk tolerance; and (iii) their information needs, uncertainties and satisfaction towards HF treatment., Methods: This qualitative patient preference study consisted of a literature review with a systematic search strategy and semi-structured interviews with HF patients, analysed using the framework method. During the interviews, patients were asked to rank a predefined list of disease and treatment-related characteristics informed by the literature review and were able to spontaneously raise additional characteristics., Results: The study included 14 Belgian HF patients (age range: 58-79, mean age: 72). (i) Regarding their unmet medical needs, HF patients reported that the most important unmet medical needs were shortness of breath and fatigue, as they negatively impact their quality of life (QoL) and independence. In the ranking exercise, patients prioritized improvements in QoL over improvements in life expectancy, whereby the following characteristics received the highest cumulative score: (1) independence, (2) shortness of breath, (3) impaired renal function, (4) survival, (5) fatigue, (6) risk of hospitalization and (7) communication with and between physicians. Patients most often spontaneously raise characteristics related to the general care process. Mechanism of action, route of administration, dose frequency and weight fluctuations scored among the least important characteristics. (ii) Regarding patients' risk tolerance towards HF treatment, some of the patients expressed zero tolerance for side effects, as they had not yet experienced any discomfort caused by the treatment or disease. (iii) Regarding their information needs, patients voiced their desire to receive practical and comprehensible advice orally from their physician because they highly value individualized treatment decision-making. Patients also expressed uncertainties regarding whether the experienced effects were due to their treatment, disease, ageing or other comorbidities., Conclusions: This study shows that, besides increasing life expectancy, HF patients prioritize improvements in symptoms and side effects reducing their QoL and independence, such as shortness of breath and fatigue. The patient-relevant characteristics identified in this study, from the perspective of HF patients themselves, may be useful to inform clinical trial endpoint selection and guide downstream drug development, evaluation and clinical decision-making towards addressing the unmet medical needs and treatment outcomes of importance to HF patients., (© 2024 The Author(s). ESC Heart Failure published by John Wiley & Sons Ltd on behalf of European Society of Cardiology.)

Glycoside Hydrolase family 65 (GH65) is a unique family of carbohydrate-active enzymes. It is the first protein family to bring together glycoside hydrolases, glycoside phosphorylases and glycosyltransferases, thereby spanning a broad range of reaction types. These enzymes catalyze the hydrolysis, reversible phosphorolysis or synthesis of various α-glucosides, typically α-glucobioses or their derivatives. In this review, we present a comprehensive overview of the diverse reaction types and substrate specificities found in family GH65. We describe the determinants that control this remarkable diversity, as well as the applications of GH65 enzymes for carbohydrate synthesis., (© 2024. The Author(s).)

Introduction: The COVID-19 pandemic required a significant response from global healthcare systems. In Belgium, the crisis began in March 2020, prompting quick action in hospitals. This study assesses the effectiveness of Belgium's hospital emergency plans and compares them with global standards for potential enhancements., Methodology: An online survey targeting CEOs of 60 Flemish general hospitals evaluated the deployment of hospital emergency coordination cells during the pandemic's first and fourth waves, utilizing various statistical analyses., Results: Findings indicate a high establishment rate of COVID-19 coordination cells before the government's deadline. Despite this readiness, differences in leadership, involvement, and communication strategies were noted among hospitals. There was a notable shift towards hybrid meetings and an evolving role for coordination cells, highlighting the need for a more structured crisis management approach., Conclusion: The study concludes that while Flemish hospitals were quick to respond, the lack of a standardized framework suggests the potential for adopting models like the Hospital Incident Command System (HICS) for improved crisis management. Future research should examine the long-term effects of these strategies and the integration of comprehensive emergency management systems in Belgium's healthcare.

Carbohydrate-active enzymes (CAZymes) can be found in all domains of life and play a crucial role in metabolic and physiological processes. CAZymes often possess a modular structure, comprising not only catalytic domains but also associated domains such as carbohydrate-binding modules (CBMs) and linker domains. By exploring the modular diversity of CAZy families, catalysts with novel properties can be discovered and further insight in their biological functions and evolutionary relationships can be obtained. Here we present the carbohydrate-active enzyme domain analysis tool (CANDy), an assembly of several novel scripts, tools and databases that allows users to analyze the domain architecture of all protein sequences in a given CAZy family. CANDy's usability is shown on glycoside hydrolase family 48, a small yet underexplored family containing multi-domain enzymes. Our analysis reveals the existence of 35 distinct domain assemblies, including eight known architectures, with the remaining assemblies awaiting characterization. Moreover, we substantiate the occurrence of horizontal gene transfer from prokaryotes to insect orthologs and provide evidence for the subsequent removal of auxiliary domains, likely through a gene fission event. CANDy is available at https://github.com/PyEED/CANDy., Competing Interests: The authors declare no potential conflict of interest., (Copyright: © 2024 Windels et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: The CBM13 family comprises carbohydrate-binding modules that occur mainly in enzymes and in several ricin-B lectins. The ricin-B lectin domain resembles the CBM13 module to a large extent. Historically, ricin-B lectins and CBM13 proteins were considered completely distinct, despite their structural and functional similarities., Results: In this data mining study, we investigate structural and functional similarities of these intertwined protein groups. Because of the high structural and functional similarities, and differences in nomenclature usage in several databases, confusion can arise. First, we demonstrate how public protein databases use different nomenclature systems to describe CBM13 modules and putative ricin-B lectin domains. We suggest the introduction of a novel CBM13 domain identifier, as well as the extension of CAZy cross-references in UniProt to guard the distinction between CAZy and non-CAZy entries in public databases. Since similar problems may occur with other lectin families and CBM families, we suggest the introduction of novel CBM InterPro domain identifiers to all existing CBM families. Second, we investigated phylogenetic, nomenclatural and structural similarities between putative ricin-B lectin domains and CBM13 modules, making use of sequence similarity networks. We concluded that the ricin-B/CBM13 superfamily may be larger than initially thought and that several putative ricin-B lectin domains may display CAZyme functionalities, although biochemical proof remains to be delivered., Conclusions: Ricin-B lectin domains and CBM13 modules are associated groups of proteins whose database semantics are currently biased towards ricin-B lectins. Revision of the CAZy cross-reference in UniProt and introduction of a dedicated CBM13 domain identifier in InterPro may resolve this issue. In addition, our analyses show that several proteins with putative ricin-B lectin domains show very strong structural similarity to CBM13 modules. Therefore ricin-B lectin domains and CBM13 modules could be considered distant members of a larger ricin-B/CBM13 superfamily., (© 2024. The Author(s).)

Despite their broad application potential, the widespread use of β-1,3-glucans has been hampered by the high cost and heterogeneity associated with current production methods. To address this challenge, scalable and economically viable processes are needed for the production of β-1,3-glucans with tailorable molecular mass distributions. Glycoside phosphorylases have shown to be promising catalysts for the bottom-up synthesis of β-1,3-(oligo)glucans since they combine strict regioselectivity with a cheap donor substrate (i.e., α-glucose 1-phosphate). However, the need for an expensive priming substrate (e.g., laminaribiose) and the tendency to produce shorter oligosaccharides still form major bottlenecks. Here, we report the discovery and application of a thermostable β-1,3-oligoglucan phosphorylase originating from Anaerolinea thermophila ( At βOGP). This enzyme combines a superior catalytic efficiency toward glucose as a priming substrate, high thermostability, and the ability to synthesize high molecular mass β-1,3-glucans up to DP 75. Coupling of At βOGP with a thermostable variant of Bifidobacterium adolescentis sucrose phosphorylase enabled the efficient production of tailorable β-1,3-(oligo)glucans from sucrose, with a near-complete conversion of >99 mol %. This cost-efficient process for the conversion of renewable bulk sugar into β-1,3-(oligo)glucans should facilitate the widespread application of these versatile functional fibers across various industries.

Glycosidic osmolytes are widespread natural compounds that protect microorganisms and their macromolecules from the deleterious effects of various environmental stresses. Their protective properties have attracted considerable interest for industrial applications, especially as active ingredients in cosmetics and healthcare products. In that regard, the osmolyte glucosylglycerate is somewhat overlooked. Glucosylglycerate is typically accumulated by certain organisms when they are exposed to high salinity and nitrogen starvation, and its potent stabilizing effects have been demonstrated in vitro. However, the applications of this osmolyte have not been thoroughly explored due to the lack of a cost-efficient production process. Here, we present an overview of the progress that has been made in developing promising strategies for the synthesis of glucosylglycerate and its precursor glycerate, and discuss the remaining challenges. KEY POINTS: • Bacterial milking could be explored for fermentative production of glucosylglycerate • Glycoside phosphorylases of GH13_18 represent attractive alternatives for biocatalytic production • Conversion of glycerol with alditol oxidase is a promising strategy for generating the precursor glycerate., (© 2024. The Author(s).)

Introduction: The goal of the Health Technology Assessment (HTA) Regulation 2021/2282 is to establish a more harmonized HTA framework, fostering member states cooperation and enabling equal patient access to innovative health technologies in Europe. This research aimed to assess the impact of the regulation on national HTAs, the strategic implications for health technology developers, and its influence on price and reimbursement negotiations. Methods: A scoping literature review encompassing peer-reviewed literature as well as grey literature was conducted. Between February and March 2023, semi-structured interviews (n = 20) were performed with stakeholders from Belgian governmental institutions, European institutions, advanced therapy medicinal product developers, academics, and sickness funds. The interviews were analyzed using the framework analysis method. Results: Numerous steps, such as the development of implementing acts and procedural guidelines remain to be taken. At member state level, national/regional HTA bodies and payers must act to adopt the new concepts of Joint Scientific Consultations (JSC) and Joint Clinical Assessments (JCA) within their national legislation, as well as revise their timelines and prepare for interactions at a European level. Compiling a harmonized PICO (Population, Intervention, Comparator, and Outcome), adapting local procedures, and increasing capacity to actively take part in the JSC and JCA are seen as primary barriers by several stakeholders. Training and education will help HTA bodies, payers, and health technology developers to participate in the European processes. Conclusion: While practical and legal challenges were identified, recommendations (such as actively preparing for the upcoming changes and increasing capacity while providing training) were provided to adapt national and European procedures to the needs of the HTA Regulation 2021/2282. The importance of fostering collaborations and aligning local HTA procedures with the new way of working set out by the Regulation was demonstrated with this study., Competing Interests: Authors MB, FV, and ST were employed by AxTalis B.V. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Desmet, Brijs, Vanderdonck, Tops, Simoens and Huys.)

Objectives: Stakeholder involvement has long been considered a success factor for a joint European health technology assessment (HTA) process, and its relevance is now anchored in the EU HTA Regulation's (EU HTAR) legislative wording. Therefore, we aimed to explore the roles, challenges, and most important activities to increase the level of involvement per stakeholder group., Methods: At the 2022 Fall Convention of the European Access Academy (EAA), working groups addressed the involvement of patients, clinicians, regulators, health technology developers (HTD), and national HTA bodies and payers within the EU HTA process. Each working group revisited the pre-convention survey results, determined key role characteristics for each stakeholder, and agreed on the most important activities to fulfill the role profile. Finally, the activities suggested per group were prioritized by plenary group., Results: The prioritized actions for patients included training and capacity building, the establishment of a patient involvement committee, and the establishment of a patient unit at the EC secretariat. For clinicians, it included alignment on evidence assessment from a clinical vs. HTA point of view, capacity building, and standardization of processes. The most important actions for regulators are to develop joint regulatory-HTA guidance documents, align processes and interfaces under the regulation, and share discussions on post-licensing evidence generation. HTDs prioritized scientific advice capacity and the review of the scoping process, and further development of the scope of the assessment report fact checks. The top three actions for national HTA bodies and payers included clarification on the early HTD dialogue process, political support and commitment, and clarification on financial support., Conclusions: Addressing the activities identified as the most important for stakeholders/collaborators in the EU HTA process (e.g., in the implementation of the EU HTA Stakeholder Network and of the guidance documents developed by the EUnetHTA 21 consortium) will be key to starting an " inclusive civil society dialogue ", as suggested by the European Commission's Pharmaceutical Strategy., Competing Interests: Conflicts of InterestT.D.: no CoI; W.V.D.: no CoI; S.S.: no CoI; I.H.: no CoI; R.G.: no CoI; M.T.: no CoI; C.D.: as a strategic and legal consultant, regularly receives honoraria for consulting from numerous health technology developers; J.D.: as a strategic and legal consultant, regularly receives honoraria for consulting from numerous health technology developers; A.C.: no CoI; F.H.: no CoI; M.P.: no CoI; M.B.: no CoI; P.M.: no CoI; A.S.: no CoI; W.G.: no CoI; F.G.: no CoI; S.C.: no CoI; J.R. (James Ryan): employed by AstraZeneca; P.D.: employed by Abbvie; O.S.-M.: no CoI., (© 2024 by the authors.)

The acylation of flavonoids serves as a means to alter their physicochemical properties, enhance their stability, and improve their bioactivity. Compared with natural flavonoid glycosides, the acylation of nonglycosylated flavonoids presents greater challenges since they contain fewer reactive sites. In this work, we propose an efficient strategy to solve this problem based on a first α-glucosylation step catalyzed by a sucrose phosphorylase, followed by acylation using a lipase. The method was applied to phloretin, a bioactive dihydrochalcone mainly present in apples. Phloretin underwent initial glucosylation at the 4'-OH position, followed by subsequent (and quantitative) acylation with C8, C12, and C16 acyl chains employing an immobilized lipase from Thermomyces lanuginosus . Electrospray ionization-mass spectrometry (ESI-MS) and two-dimensional nuclear magnetic resonance spectroscopy (2D-NMR) confirmed that the acylation took place at 6-OH of glucose. The water solubility of C8 acyl glucoside closely resembled that of aglycone, but for C12 and C16 derivatives, it was approximately 3 times lower. Compared with phloretin, the radical scavenging capacity of the new derivatives slightly decreased with 2,2-diphenyl-1-picrylhydrazyl (DPPH) and was similar to 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS •+ ). Interestingly, C12 acyl-α-glucoside displayed an enhanced (3-fold) transdermal absorption (using pig skin biopsies) compared to phloretin and its α-glucoside.

The reconstruction of ancestral sequences can offer a glimpse into the fascinating process of molecular evolution by exposing the adaptive pathways that shape the proteins found in nature today. Here, we track the evolution of the carbohydrate-active enzymes responsible for the synthesis and turnover of mannogen, a critical carbohydrate reserve in Leishmania parasites. Biochemical characterization of resurrected enzymes demonstrated that mannoside phosphorylase activity emerged in an ancestral bacterial mannosyltransferase, and later disappeared in the process of horizontal gene transfer and gene duplication in Leishmania . By shuffling through plausible historical sequence space in an ancestral mannosyltransferase, we found that mannoside phosphorylase activity could be toggled on through various combinations of mutations at positions outside of the active site. Molecular dynamics simulations showed that such mutations can affect loop rigidity and shield the active site from water molecules that disrupt key interactions, allowing α-mannose 1-phosphate to adopt a catalytically productive conformation. These findings highlight the importance of subtle distal mutations in protein evolution and suggest that the vast collection of natural glycosyltransferases may be a promising source of engineering templates for the design of tailored phosphorylases., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

Background: SARS-CoV-2-neutralizing antibodies (nABs) showed great promise in the early phases of the COVID-19 pandemic. The emergence of resistant strains, however, quickly rendered the majority of clinically approved nABs ineffective. This underscored the imperative to develop nAB cocktails targeting non-overlapping epitopes., Methods: Undertaking a nAB discovery program, we employed a classical workflow, while integrating artificial intelligence (AI)-based prediction to select non-competing nABs very early in the pipeline. We identified and in vivo validated (in female Syrian hamsters) two highly potent nABs., Findings: Despite the promising results, in depth cryo-EM structural analysis demonstrated that the AI-based prediction employed with the intention to ensure non-overlapping epitopes was inaccurate. The two nABs in fact bound to the same receptor-binding epitope in a remarkably similar manner., Interpretation: Our findings indicate that, even in the Alphafold era, AI-based predictions of paratope-epitope interactions are rough and experimental validation of epitopes remains an essential cornerstone of a successful nAB lead selection., Funding: Full list of funders is provided at the end of the manuscript., Competing Interests: Declaration of interests Ghent University has filed for patent protection on the antibody sequences described herein, and D.D.A., M.W., R.W., W.W., S.G. and L.V. are named as co-inventors on this patent (European Patent Application: 21186206.5). A.P. is employee of the MAbSilico, H.R. holds a patent regarding neutralizing VHH antibodies binding the Spike RBD (PCT/EP2021/052885) and has filed a priority application for neutralizing VHH antibodies binding Spike S2 (EP 23160838.1). X.S. is a recipient of FWO research project COVID-19 (G0G4920N) and FWO-FNRS project VIREOS (EOS ID: 30981113) grants., (Copyright © 2023. Published by Elsevier B.V.)