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Abstract: An in silico survey of all known 3D-structures of glycoside hydrolases that contain a ligand in the −1 subsite is presented. A recurrent crucial positioning of active site residues indicates a common general strategy for electrostatic stabilisation directed to the carbohydrate’s ring-oxygen at the transition state. This is substantially different depending on whether the enzyme’s proton donor is syn or anti positioned versus the substrate. A comprehensive list of enzymes belonging to 42 different families is given and selected examples are described. An implication for an early evolution scenario of glycoside hydrolases is discussed. [Copyright &y& Elsevier]

An in silico survey of the −1 subsite of all known 3D-structures of O-glycoside hydrolases containing a suitably positioned ligand has led to the recognition – apparently without exceptions – of a transition state stabilising hydrophobic platform which is complementary to a crucial hydrophobic patch of the ligand. This platform is family-specific and highly conserved. A comprehensive list is given with examples of enzymes belonging to 33 different families. Several typical constellations of platform – protein residues are described. [Copyright &y& Elsevier]

The degree to which molecular epidemiology reveals information about the sources and transmission patterns of an outbreak depends on the resolution of the technology used and the samples studied. Isolates of Escherichia coli O104:H4 from the outbreak centered in Germany in May–July 2011, and the much smaller outbreak in southwest France in June 2011, were indistinguishable by standard tests. We report a molecular epidemiological analysis using multiplatform whole-genome sequencing and analysis of multiple isolates from the German and French outbreaks. Isolates from the German outbreak showed remarkably little diversity, with only two single nucleotide polymorphisms (SNPs) found in isolates from four individuals. Surprisingly, we found much greater diversity (19 SNPs) in isolates from seven individuals infected in the French outbreak. The German isolates form a clade within the more diverse French outbreak strains. Moreover, five isolates derived from a single infected individual from the French outbreak had extremely limited diversity. The striking difference in diversity between the German and French outbreak samples is consistent with several hypotheses, including a bottleneck that purged diversity in the German isolates, variation in mutation rates in the two E. coli outbreak populations, or uneven distribution of diversity in the seed populations that led to each outbreak.

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.)

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.)

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).)

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.)

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.)

Involvement of all relevant stakeholders will be of utmost importance for the success of the developing EU HTA harmonization process. A multi-step procedure was applied to develop a survey across stakeholders/collaborators within the EU HTA framework to assess their current level of involvement, determine their suggested future role, identify challenges to contribution, and highlight efficient ways to fulfilling their role. The 'key' stakeholder groups identified and covered by this research included: patients', clinicians', regulatory, and Health Technology Developer representatives. The survey was circulated to a wide expert audience including all relevant stakeholder groups in order to determine self-perception by the 'key' stakeholders regarding involvement in the HTA process (self-rating), and in a second, slightly modified version of the questionnaire, to determine the perception of 'key' stakeholder involvement by HTA bodies, payers, and policymakers (external rating). Predefined analyses were conducted on the submitted responses. Fifty-four responses were received (patients 9; clinicians: 8; regulators: 4; HTDs 14; HTA bodies: 7; Payers: 5; policymakers 3; others 4). The mean self-perceived involvement score was consistently lower for each of the 'key' stakeholder groups than the respective external ratings. Based on the qualitative insights generated in the survey, a RACI Chart (Responsible/Accountable/Consulted/Informed) was developed for each of the stakeholder groups to determine their roles and involvement in the current EU HTA process. Our findings suggest extensive effort and a distinct research agenda are required to ensure adequate involvement of the key stakeholder groups in the evolving EU HTA process., Competing Interests: The views expressed in this article are the personal views of the author(s) and may not be understood or quoted as being made on behalf of or reflecting the position of the regulatory agency/agencies or organizations with which the author(s) is/are employed/affiliated., (© 2023 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.)

Rare sugars have recently attracted attention as potential sugar replacers. Understanding the biochemical and biological behavior of these sugars is of importance in (novel) food formulations and prevention of type 2 diabetes. In this study, we investigated whether rare sugars may positively affect intestinal and liver metabolism, as well as muscle insulin sensitivity, compared to conventional sugars. Rare disaccharide digestibility, hepatic metabolism of monosaccharides (respirometry) and the effects of sugars on skeletal muscle insulin sensitivity (impaired glucose uptake) were investigated in, respectively, Caco-2, HepG2 and L6 cells or a triple coculture model with these cells. Glucose and fructose, but not l-arabinose, acutely increased extracellular acidification rate (ECAR) responses in HepG2 cells and impaired glucose uptake in L6 cells following a 24 h exposure at 28 mM. Cellular bioenergetics and digestion experiments with Caco-2 cells indicate that especially trehalose (α1-1α), D-Glc-α1,2-D-Gal, D-Glc-α1,2-D-Rib and D-Glc-α1,3-L-Ara experience delayed digestion and reduced cellular impact compared to maltose (α1-4), without differences on insulin-stimulated glucose uptake in a short-term setup with a Caco-2/HepG2/L6 triple coculture. These results suggest a potential for l-arabinose and specific rare disaccharides to improve metabolic health; however, additional in vivo research with longer sugar exposures should confirm their beneficial impact on insulin sensitivity in humans.

The commercial value of specialty carbohydrates and glycosylated compounds has sparked considerable interest in the synthetic potential of carbohydrate-active enzymes (CAZymes). Protein engineering methods have proven to be highly successful in expanding the range of glycosylation reactions that these enzymes can perform efficiently and cost-effectively. The past few years have witnessed meaningful progress in this area, largely due to a sharper focus on the understanding of structure-function relationships and mechanistic intricacies. Here, we summarize recent studies that demonstrate how protein engineers have become much better at traversing the fitness landscape of CAZymes through mutational bridges that connect the different activity types., (Copyright © 2022. Published by Elsevier Ltd.)

Protein evolution or engineering studies are traditionally focused on amino acid substitutions and the way these contribute to fitness. Meanwhile, the insertion and deletion of amino acids is often overlooked, despite being one of the most common sources of genetic variation. Recent methodological advances and successful engineering stories have demonstrated that the time is ripe for greater emphasis on these mutations and their understudied effects. This review highlights the evolutionary importance and biotechnological relevance of insertions and deletions (indels). We provide a comprehensive overview of approaches that can be employed to include indels in random, (semi)-rational or computational protein engineering pipelines. Furthermore, we discuss the tolerance to indels at the structural level, address how domain indels can link the function of unrelated proteins, and feature studies that illustrate the surprising and intriguing potential of frameshift mutations., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Countries are struggling to provide affordable access to medicines while supporting the market entry of innovative, expensive products. This Perspective aims to discuss challenges and avenues for balancing health care system objectives of access, affordability and innovation related to medicines in Belgium (and in other countries). Methods: This Perspective focuses on the R&D, regulatory approval and market access phases, with particular attention to oncology medicines, precision medicines, orphan medicines, advanced therapies, repurposed medicines, generics and biosimilars. The authors conducted a narrative review of the peer-reviewed literature, of the grey literature (such as policy documents and reports of consultancy agencies), and of their own research. Results: Health care stakeholders need to consider various initiatives for balancing innovation with access to medicines, which relate to clinical and non-clinical outcomes (e.g. supporting the conduct of pragmatic clinical trials, treatment optimisation and patient preference studies, optimising the use of real-world evidence in market access decision making), value assessment (e.g. increasing the transparency of the reimbursement system and criteria, tailoring the design of managed entry agreements to specific types of uncertainty), affordability (e.g. harnessing the role of generics and biosimilars in encouraging price competition, maximising opportunities for personalising and repurposing medicines) and access mechanisms (e.g. promoting collaboration and early dialogue between stakeholders including patients). Conclusion : Although there is no silver bullet that can balance valuable innovation with affordable access to medicines, (Belgian) policy and decision makers should continue to explore initiatives that exploit the potential of both the on-patent and off-patent pharmaceutical markets., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Simoens, Abdallah, Barbier, Lacosta, Blonda, Car, Claessens, Desmet, De Sutter, Govaerts, Janssens, Lalova, Moorkens, Saesen, Schoefs, Vandenplas, Van Overbeeke, Verbaanderd and Huys.)

Nucleotide sugar (NS) dehydratases play a central role in the biosynthesis of deoxy and amino sugars, which are involved in a variety of biological functions in all domains of life. Bacteria are true masters of deoxy sugar biosynthesis as they can produce a wide range of highly specialized monosaccharides. Indeed, deoxy and amino sugars play important roles in the virulence of gram-positive and gram-negative pathogenic species and are additionally involved in the biosynthesis of diverse macrolide antibiotics. The biosynthesis of deoxy sugars relies on the activity of NS dehydratases, which can be subdivided into three groups based on their structure and reaction mechanism. The best-characterized NS dehydratases are the 4,6-dehydratases that, together with the 5,6-dehydratases, belong to the NS-short-chain dehydrogenase/reductase superfamily. The other two groups are the less abundant 2,3-dehydratases that belong to the Nudix hydrolase superfamily and 3-dehydratases, which are related to aspartame aminotransferases. 4,6-Dehydratases catalyze the first step in all deoxy sugar biosynthesis pathways, converting nucleoside diphosphate hexoses to nucleoside diphosphate-4-keto-6-deoxy hexoses, which in turn are further deoxygenated by the 2,3- and 3-dehydratases to form dideoxy and trideoxy sugars. In this review, we give an overview of the NS dehydratases focusing on the comparison of their structure and reaction mechanisms, thereby highlighting common features, and investigating differences between closely related members of the same superfamilies., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Non-alcoholic fatty liver disease (NAFLD) has become the most prevalent liver disease worldwide and is impacted by an unhealthy diet with excessive calories, although the role of sugars in NAFLD etiology remains largely unexplored. Rare sugars are natural sugars with alternative monomers and glycosidic bonds, which have attracted attention as sugar replacers due to developments in enzyme engineering and hence an increased availability. We studied the impact of (rare) sugars on energy production, liver cell physiology and gene expression in human intestinal colorectal adenocarcinoma (Caco-2) cells, hepatoma G2 (HepG2) liver cells and a coculture model with these cells. Fat accumulation was investigated in the presence of an oleic/palmitic acid mixture. Glucose, fructose and galactose, but not mannose, l-arabinose, xylose and ribose enhanced hepatic fat accumulation in a HepG2 monoculture. In the coculture model, there was a non-significant trend ( p = 0.08) towards higher (20-55% increased) median fat accumulation with maltose, kojibiose and nigerose. In this coculture model, cellular energy production was increased by glucose, maltose, kojibiose and nigerose, but not by trehalose. Furthermore, glucose, fructose and l-arabinose affected gene expression in a sugar-specific way in coculture HepG2 cells. These findings indicate that sugars provide structure-specific effects on cellular energy production, hepatic fat accumulation and gene expression, suggesting a health potential for trehalose and l-arabinose, as well as a differential impact of sugars beyond the distinction of conventional and rare sugars.

GDP-mannose 3,5-epimerase (GM35E, GME) belongs to the short-chain dehydrogenase/reductase (SDR) protein superfamily and catalyses the conversion of GDP-d-mannose towards GDP-l-galactose. Although the overall reaction seems relatively simple (a double epimerization), the enzyme needs to orchestrate a complex set of chemical reactions, with no less than 6 catalysis steps (oxidation, 2x deprotonation, 2x protonation and reduction), to perform the double epimerization of GDP-mannose to GDP-l-galactose. The enzyme is involved in the biosynthesis of vitamin C in plants and lipopolysaccharide synthesis in bacteria. In this review, we provide a clear overview of these interesting epimerases, including the latest findings such as the recently characterized bacterial and thermostable GM35E representative and its mechanism revision but also focus on their industrial potential in rare sugar synthesis and glycorandomization., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Beerens, Gevaert and Desmet.)

The Glycoside Hydrolase Family 65 (GH65) is an enzyme family of inverting α-glucoside phosphorylases and hydrolases that currently contains 10 characterized enzyme specificities. However, its sequence diversity has never been studied in detail. Here, an in-silico analysis of correlated mutations was performed, revealing specificity-determining positions that facilitate annotation of the family's phylogenetic tree. By searching these positions for amino acid motifs that do not match those found in previously characterized enzymes from GH65, several clades that may harbor new functions could be identified. Three enzymes from across these regions were expressed in E. coli and their substrate profile was mapped. One of those enzymes, originating from the bacterium Mucilaginibacter mallensis , was found to hydrolyze kojibiose and α-1,2-oligoglucans with high specificity. We propose kojibiose glucohydrolase as the systematic name and kojibiose hydrolase or kojibiase as the short name for this new enzyme. This work illustrates a convenient strategy for mapping the natural diversity of enzyme families and smartly mining the ever-growing number of available sequences in the quest for novel specificities.

Short-chain Dehydrogenase/Reductase enzymes that are active on nucleotide sugars (abbreviated as NS-SDR) are of paramount importance in the biosynthesis of rare sugars and glycosides. Some family members have already been extensively characterized due to their direct implication in metabolic disorders or in the biosynthesis of virulence factors. In this review, we combine the knowledge gathered from studies that typically focused only on one NS-SDR activity with an in-depth analysis and overview of all of the different NS-SDR families (169,076 enzyme sequences). Through this structure-based multiple sequence alignment of NS-SDRs retrieved from public databases, we could identify clear patterns in conservation and correlation of crucial residues. Supported by this analysis, we suggest updating and extending the UDP-galactose 4-epimerase "hexagonal box model" to an "heptagonal box model" for all NS-SDR enzymes. This specificity model consists of seven conserved regions surrounding the NDP-sugar substrate that serve as fingerprint for each specificity. The specificity fingerprints highlighted in this review will be beneficial for functional annotation of the large group of NS-SDR enzymes and form a guide for future enzyme engineering efforts focused on the biosynthesis of rare and specialty carbohydrates., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Genetic studies in underrepresented populations identify disproportionate numbers of novel associations. However, most genetic studies use genotyping arrays and sequenced reference panels that best capture variation most common in European ancestry populations. To compare data generation strategies best suited for underrepresented populations, we sequenced the whole genomes of 91 individuals to high coverage as part of the Neuropsychiatric Genetics of African Population-Psychosis (NeuroGAP-Psychosis) study with participants from Ethiopia, Kenya, South Africa, and Uganda. We used a downsampling approach to evaluate the quality of two cost-effective data generation strategies, GWAS arrays versus low-coverage sequencing, by calculating the concordance of imputed variants from these technologies with those from deep whole-genome sequencing data. We show that low-coverage sequencing at a depth of ≥4× captures variants of all frequencies more accurately than all commonly used GWAS arrays investigated and at a comparable cost. Lower depths of sequencing (0.5-1×) performed comparably to commonly used low-density GWAS arrays. Low-coverage sequencing is also sensitive to novel variation; 4× sequencing detects 45% of singletons and 95% of common variants identified in high-coverage African whole genomes. Low-coverage sequencing approaches surmount the problems induced by the ascertainment of common genotyping arrays, effectively identify novel variation particularly in underrepresented populations, and present opportunities to enhance variant discovery at a cost similar to traditional approaches., (Copyright © 2021 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Epimerization of sugar nucleotides is central to the structural diversification of monosaccharide building blocks for cellular biosynthesis. Epimerase applicability to carbohydrate synthesis can be limited, however, by the high degree of substrate specificity exhibited by most sugar nucleotide epimerases. Here, we discovered a promiscuous type of CDP-tyvelose 2-epimerase (TyvE)-like enzyme that promotes C2-epimerization in all nucleotide (CDP, UDP, GDP, ADP, TDP)-activated forms of d-glucose. This new epimerase, originating from Thermodesulfatator atlanticus , is a functional homodimer that contains one tightly bound NAD + /subunit and shows optimum activity at 70°C and pH 9.5. The enzyme exhibits a k cat with CDP-dglucose of ∼1.0 min -1 (pH 7.5, 60°C). To characterize the epimerase kinetically and probe its substrate specificity, we developed chemo-enzymatic syntheses for CDP-dmannose, CDP-6-deoxy-dglucose, CDP-3-deoxy-dglucose and CDP-6-deoxy- dxylo -hexopyranos-4-ulose. Attempts to obtain CDP-dparatose and CDP-dtyvelose were not successful. Using high-resolution carbohydrate analytics and in situ NMR to monitor the enzymatic conversions (60°C, pH 7.5), we show that the CDP-dmannose/CDP-dglucose ratio at equilibrium is 0.67 (± 0.1), determined from the kinetic Haldane relationship and directly from the reaction. We further show that deoxygenation at sugar C6 enhances the enzyme activity 5-fold compared to CDP-dglucose whereas deoxygenation at C3 renders the substrate inactive. Phylogenetic analysis places the T. atlanticus epimerase into a distinct subgroup within the sugar nucleotide epimerase family of SDR (short-chain dehydrogenases/reductases), for which the current study now provides the functional context. Collectively, our results expand an emerging toolbox of epimerase-catalyzed reactions for sugar nucleotide synthesis. IMPORTANCE Epimerases of the sugar nucleotide-modifying class of enzymes have attracted considerable interest in carbohydrate (bio)chemistry, for the mechanistic challenges and the opportunities for synthesis involved in the reactions catalyzed. Discovery of new epimerases with expanded scope of sugar nucleotide substrates used is important to promote the mechanistic inquiry and can facilitate the development of new enzyme applications. Here, a CDP-tyvelose 2-epimerase-like enzyme from Thermodesulfatator atlanticus is shown to catalyze sugar C2 epimerization in CDP-glucose and other nucleotide-activated forms of dglucose. The reactions are new to nature in the context of enzymatic sugar nucleotide modification. The current study explores the substrate scope of the discovered C2-epimerase and, based on modeling, suggests structure-function relationships that may be important for specificity and catalysis., (Copyright © 2020 Rapp et al.)

Background: Early 2020, a COVID-19 epidemic became a public health emergency of international concern. To address this pandemic broad testing with an easy, comfortable and reliable testing method is of utmost concern. Nasopharyngeal (NP) swab sampling is the reference method though hampered by international supply shortages. A new oropharyngeal/nasal (OP/N) sampling method was investigated using the more readily available throat swab., Results: 35 patients were diagnosed with SARS-CoV-2 by means of either NP or OP/N sampling. The paired swabs were both positive in 31 patients. The one patient who tested negative on both NP and OP/N swab on admission, was ultimately diagnosed on bronchoalveolar lavage fluid. A strong correlation was found between the viral RNA loads of the paired swabs (r = 0.76; P < 0.05). The sensitivity of NP and OP/N analysis in hospitalized patients (n = 28) was 89.3% and 92.7% respectively., Conclusions: This study demonstrates equivalence of NP and OP/N sampling for detection of SARS-CoV-2 by means of rRT-PCR. Sensitivity of both NP and OP/N sampling is very high in hospitalized patients.

Objectives: Disulfiram is an adjunct in the treatment of alcohol use disorders, but case reports indicate that disulfiram ethanol reactions are not always recognized in the emergency department. Our first aim is to remind of this risk with two case reports of life-threatening reactions not immediately considered by the emergency physician. The second aim is to estimate the probability that a disulfiram reaction goes unrecognized with the use of a retrospective study of patients admitted to the emergency department., Methods: Clinical files of patients admitted between October 1, 2010 and September 30, 2014 to the emergency department were retrospectively screened for the key words "ethanol use" and "disulfiram". Their diagnoses were then scored by a panel regarding the probability of an interaction., Results: Seventy-nine patients were included, and a disulfiram-ethanol reaction was scored as either 'highly likely', 'likely' or 'possible' in 54.4% and as 'doubtful' or 'certainly not present' in 45.6% of the patients. The interrater agreement was 0.71 (95% CI: 0.64-0.79). The diagnosis was not considered or only after a delay in 44.2% of the patients with a 'possible' to 'highly likely' disulfiram interaction. One patient with a disulfiram overdose died and was considered as a 'possible' interaction., Discussion and Conclusions: A disulfiram ethanol interaction can be life threatening and failure to consider the diagnosis in the emergency department seems frequent. Prospective studies with documentation of the intake of disulfiram and evaluation of the value of acetaldehyde as a biomarker are needed to determine the precise incidence. Improving knowledge of disulfiram interactions and adequate history taking of disulfiram intake may improve the care for patients., Competing Interests: The authors have declared that no competing interests exist.

Although trehalose has recently gained interest because of its pharmaceutical potential, its clinical use is hampered due to its low bioavailability. Hence, hydrolysis-resistant trehalose analogues retaining biological activity could be of interest. In this study, 34 4- and 6- O -substituted trehalose derivatives were synthesised using an ether- or carbamate-type linkage. Their hydrolysis susceptibility and inhibitory properties were determined against two trehalases, i.e. porcine kidney and Mycobacterium smegmatis . With the exception of three weakly hydrolysable 6- O -alkyl derivatives, the compounds generally showed to be completely resistant. Moreover, a number of derivatives was shown to be an inhibitor of one or both of these trehalases. For the strongest inhibitors of porcine kidney trehalase IC 50 values of around 10 mM could be determined, whereas several compounds displayed sub-mM IC 50 against M. smegmatis trehalase. Dockings studies were performed to explain the observed influence of the substitution pattern on the inhibitory activity towards porcine kidney trehalase.

Isomerization reactions are fundamental in biology. Lactate racemase, which isomerizes L- and D-lactate, is composed of the LarA protein and a nickel-containing cofactor, the nickel-pincer nucleotide (NPN). In this study, we show that LarA is part of a superfamily containing many different enzymes. We overexpressed and purified 13 lactate racemase homologs, incorporated the NPN cofactor, and assayed the isomerization of different substrates guided by gene context analysis. We discovered two malate racemases, one phenyllactate racemase, one α-hydroxyglutarate racemase, two D-gluconate 2-epimerases, and one short-chain aliphatic α-hydroxyacid racemase among the tested enzymes. We solved the structure of a malate racemase apoprotein and used it, along with the previously described structures of lactate racemase holoprotein and D-gluconate epimerase apoprotein, to identify key residues involved in substrate binding. This study demonstrates that the NPN cofactor is used by a diverse superfamily of α-hydroxyacid racemases and epimerases, widely expanding the scope of NPN-dependent enzymes.

This study compares the metabolic properties of kojibiose, trehalose, sucrose, and xylitol upon incubation with representative oral bacteria as monocultures or synthetic communities or with human salivary bacteria in a defined medium. Compared to sucrose and trehalose, kojibiose resisted metabolism during a 48-h incubation with monocultures, except for Actinomyces viscosus Incubations with Lactobacillus -based communities, as well as salivary bacteria, displayed kojibiose metabolism, yet to a lesser extent than sucrose and trehalose. Concurring with our in vitro findings, screening for carbohydrate-active enzymes revealed that only Lactobacillus spp. and A. viscosus possess enzymes from glycohydrolase (GH) families GH65 and GH15, respectively, which are associated with kojibiose metabolism. Donor-dependent differences in salivary microbiome composition were noted, and differences in pH drop during incubation indicated different rates of sugar metabolism. However, functional analysis indicated that lactate, acetate, and formate evenly dominated the metabolic profile for all sugars except for xylitol. 16S rRNA gene sequencing analysis and α-diversity markers revealed that a significant shift of the microbiome community by sugars was more pronounced in sucrose and trehalose than in kojibiose and xylitol. In Streptococcus spp., a taxon linked to cariogenesis dominated in sucrose (mean ± standard deviation, 91.8 ± 6.4%) and trehalose (55.9 ± 38.6%), representing a high diversity loss. In contrast, Streptococcus (5.1 ± 3.7%) was less abundant in kojibiose, which instead was dominated by Veillonella (26.8 ± 19.6%), while for xylitol, Neisseria (29.4 ± 19.1%) was most abundant. Overall, kojibiose and xylitol incubations stimulated cariogenic species less yet closely maintained an abundance of key phyla and genera of the salivary microbiome, suggesting that kojibiose has low cariogenic properties. IMPORTANCE This study provides a detailed scientific insight on the metabolism of a rare disaccharide, kojibiose, whose mass production has recently been made possible. While the resistance of kojibiose was established with monocultures, delayed utilization of kojibiose was observed with communities containing lactobacilli and A. viscosus as well as with complex communities of bacteria from human saliva. Kojibiose is, therefore, less metabolizable than sucrose and trehalose. Moreover, although conventional sugars cause distinct shifts in salivary microbial communities, our study has revealed that kojibiose is able to closely maintain the salivary microbiome composition, suggesting its low cariogenic properties. This study furthermore underscores the importance and relevance of microbial culture and ex vivo mixed cultures to study cariogenicity and substrate utilization; this is in sharp contrast with tests that solely rely on monocultures such as Streptococcus mutans , which clearly fail to capture complex interactions between oral microbiota., (Copyright © 2020 American Society for Microbiology.)

Sucrose phosphorylases are carbohydrate-active enzymes with outstanding potential for the biocatalytic conversion of common table sugar into products with attractive properties. They belong to the glycoside hydrolase family GH13, where they are found in subfamily 18. In bacteria, these enzymes catalyse the phosphorolysis of sucrose to yield α-glucose 1-phosphate and fructose. However, sucrose phosphorylases can also be applied as versatile transglucosylases for the synthesis of valuable glycosides and sugars because their broad promiscuity allows them to transfer the glucosyl group of sucrose to a diverse collection of compounds other than phosphate. Numerous process and enzyme engineering studies have expanded the range of possible applications of sucrose phosphorylases ever further. Moreover, it has recently been discovered that family GH13 also contains a few novel phosphorylases that are specialised in the phosphorolysis of sucrose 6 F -phosphate, glucosylglycerol or glucosylglycerate. In this review, we provide an overview of the progress that has been made in our understanding and exploitation of sucrose phosphorylases and related enzymes over the past ten years.

The monoterpene thymol has been proposed as a valuable alternative to in-feed antibiotics in animal production. However, the effectiveness of the antimicrobial is comprised by its fast absorption in the upper gastrointestinal tract. In this work, two glucoconjugates, thymol α-d-glucopyranoside (TαG) and thymol β-d-glucopyranoside (TβG), were compared with free thymol for their potential to deliver higher concentrations of the active compound to the distal small intestine of supplemented piglets. Additionally, an analytical method was developed and validated for the simultaneous quantification of thymol and its glucoconjugates in different matrices. In stomach contents of pigs fed with 3333 μmol kg -1 thymol, TαG, or TβG, total thymol concentrations amounted to 3048, 2357, and 1820 μmol kg -1 dry matter, respectively. In glucoconjugate-fed pigs, over 30% of this concentration was present in the unconjugated form, suggesting partial hydrolysis in the stomach. No quantifiable levels of thymol or glucoconjugates were detected in the small intestine or cecum for any treatment, indicating that conjugation with one glucose unit did not sufficiently protect thymol from early absorption., Competing Interests: The authors declare no competing financial interest., (Copyright © 2020 American Chemical Society.)

The present study evaluated gluco-conjugation as a measure to delay thymol absorption and enhance its antimicrobial activity in the gut of weaned piglets. The three dietary treatments consisted of a basal diet without additives (T CON ), supplemented with thymol at 3.7 mmol/kg dry matter (T THY ), or with an equimolar amount of thymol α-D-glucopyranoside (T TαG ). Each dietary treatment was replicated in 6 pens with 2 piglets per pen ( n = 12 for analytical parameters) and was supplemented for 14 days. The total (free plus gluco-conjugated) thymol concentrations in the stomach contents were 14% lower in T TαG as compared to T THY piglets. Neither of the additives could be detected further down the gut. E.coli counts in the proximal small intestine were significantly lower in T THY than in T TαG pigs (3.35 vs. 4.29 log 10 CFU/g); however, other bacterial counts and their metabolites were unaffected by treatment. A metagenomic bacterial analysis revealed a great relative abundance of Lactobacillus spp. in the distal small intestine (range 88.4%-99.9%), irrespective of treatment. The intestinal barrier function was improved by T THY , but not T TαG , compared to T CON. In conclusion, gluco-conjugation did not result in higher thymol concentrations in the gut, but conversely, it seemed to diminish the biological effects of thymol in vivo., Competing Interests: 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.