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Introduction: Respiratory syncytial virus is a leading cause of respiratory hospitalisations in infants. This systematic review (registration number: CRD42021248309) aims to synthesise the available evidence on Respiratory Syncytial Virus-related hospitalisations among children aged 0 to 6 years in Italy., Methods: The literature search was conducted on PubMed, Embase, Scopus, and International HTA, covering the period from January 2000 to July 2022, with a focus on studies that reported information on Respiratory Syncytial Virus-associated hospitalisation in children aged 0-6 years in Italy., Results: Eight articles were included after screening 20,845 records. These retrospective studies reported that most hospitalisations were among those <1 year (71.5%-88.8%), infants aged <1 year were also at higher risk of hospitalisation in intensive care unit. Respiratory Syncytial Virus infections typically peaked December-February, with an atypical early start in August 2021. Subtype analysis showed alternating prevalence of Respiratory Syncytial Virus-A and Respiratory Syncytial Virus-B across different seasons. Coinfections were not uncommon (1.1%-37.4%), with rhinovirus and bocavirus being the most frequent., Conclusions: All infants at their first Respiratory Syncytial Virus season showed an increased risk of severe infection and hospitalisation, regardless of the gestational age at birth, compared to older participants. This systematic review will enrich the understanding about Respiratory Syncytial Virus disease and help support decisions regarding prevention efforts in Italy.

Introduction: Despite global immunization efforts, rubella remains a public health concern, particularly in high- and middle-income countries. This study focused on rubella seroprevalence in the province of Florence, Italy, aiming to identify susceptibility clusters, especially among women in their childbearing age., Methods: A cross-sectional study was conducted between April 2018 and December 2019, enrolling 430 adult subjects (age over 18 years). Serum samples were collected, and anti-rubella antibodies were quantified using the ELISA test. Data were analyzed descriptively and compared by sex, nationality, and age groups using statistical tests., Results: The overall rubella seroprevalence was high (92.3%), with no significant differences between genders or nationalities. Among childbearing-age females (18-49 years), the highest seroprevalence was observed in the 30-39 age group (94.1%). However, susceptibility clusters exceeding the 5% threshold set by WHO were identified, especially in females aged 40-49 years (7.0%)., Conclusions: Despite high overall seroprevalence, the study identified pockets of susceptibility, even in childbearing age women. Continuous monitoring, targeted immunization strategies, and public health interventions are recommended to maintain rubella elimination, emphasizing the importance of sustained vaccination efforts to protect vulnerable populations.

Hyaluronan (hyaluronic acid, HA), a key glycosaminoglycan in the extracellular matrix, plays crucial roles in various physiological and pathological processes, including development, tissue hydration, inflammation, and tumor progression. Traditional methods for HA quantification, such as ELISA-like assays, often have limitations in sensitivity and specificity, particularly for lower molecular weight HA. In this work, we introduce a coupled liquid chromatographic-tandem mass spectrometric (LC-MS/MS) method that employs a chemoenzymatically synthesized 13 C-labeled lyase-derived authentic HA disaccharide calibrant for quantification of HA at the nanogram level. The method was validated against three HA polysaccharides with the sizes of ~33, 210, and 540 kDa. We applied this quantification technique to mouse tissues and plasma from both healthy and acetaminophen-induced acute liver injury mice. Our data revealed a ~75-fold increase in HA concentration in the liver of acetaminophen-injured mice with a concomitant depletion from plasma. Overall, our method offers a robust, universal, and highly sensitive tool for HA analysis in diverse biological samples that will advance the investigation of the roles of this polysaccharide in human disease conditions., Competing Interests: J.L. is a founder and the chief scientific officer for Glycan Therapeutics. K.A. is a founder of Glyco Discoveries, a subsidiary of Glycan Therapeutics. Both J.L. and K.A. have equity in Glycan Therapeutics. P.L.D. has commercialized the quasi‐monodisperse HA reagent production and receives financial compensation for Hyalose, LLC products licensed by Echelon Biosciences, Inc. The other authors declare no conflicts of interest., (© 2024 The Author(s). Proteoglycan Research published by Wiley Periodicals LLC.)

Despite the recent progress on the solution-phase enzymatic synthesis of heparan sulfate (HS) and chondroitin sulfate (CS), solid-phase enzymatic synthesis has not been fully investigated. Here, we describe the solid-phase enzymatic synthesis of HS and CS backbone oligosaccharides using specialized linkers. We demonstrate the use of immobilized HS linker to synthesize CS, and the use of immobilized CS linker to synthesize HS. The linkers were then digested with chondroitin ABCase and heparin lyases, respectively, to obtain the products. Our findings uncover a potential approach for accelerating the synthesis of structurally homogeneous HS and CS oligosaccharides., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Objectives: Respiratory syncytial virus (RSV) is a frequent cause of acute lower respiratory infection in children, imposing a substantial economic burden on healthcare systems. This systematic review aimed to assess the economic burden and healthcare utilisation of RSV in children aged 0-59 months in Italy., Study Design: Systematic review., Methods: A systematic search of PubMed, Embase, Scopus, and the International HTA Database, including studies published in English or Italian, was conducted between January 2000 and July 2022. Inclusion criteria required studies to be conducted in Italy and provide data on the economic costs and healthcare resource utilisation related to RSV infections., Results: Out of 20,845 records screened, 18 articles met the inclusion criteria. Only one study provided comprehensive data on RSV disease costs, including hospitalisation, diagnostic tests, and medical procedures for infants with RSV-bronchiolitis. The mean cost per inpatient was higher for RSV-positive children (€5753.43 ± €2041.62) than that for RSV-negative children. Additionally, five studies reported a median length of hospital stay of 5 days for RSV-infected children, and four studies indicated a higher frequency of intensive care unit admissions for RSV-infected children than for those with other viral infections., Conclusions: This is the first systematic review to examine the economic burden and healthcare utilisation of RSV in children aged 0-59 months in Italy. While limited data were available, the findings underscore the urgency to conduct further research and gather additional evidence on the costs and healthcare resource utilisation associated with RSV infections. Such efforts are essential for informing the development of effective prevention strategies for paediatric RSV infections in Italy., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

SARS-CoV-2 infection causes spike-dependent fusion of infected cells with ACE2 positive neighboring cells, generating multi-nuclear syncytia that are often associated with severe COVID. To better elucidate the mechanism of spike-induced syncytium formation, we combine chemical genetics with 4D confocal imaging to establish the cell surface heparan sulfate (HS) as a critical stimulator for spike-induced cell-cell fusion. We show that HS binds spike and promotes spike-induced ACE2 clustering, forming synapse-like cell-cell contacts that facilitate fusion pore formation between ACE2-expresing and spike-transfected human cells. Chemical or genetic inhibition of HS mitigates ACE2 clustering, and thus, syncytium formation, whereas in a cell-free system comprising purified HS and lipid-anchored ACE2, HS stimulates ACE2 clustering directly in the presence of spike. Furthermore, HS-stimulated syncytium formation and receptor clustering require a conserved ACE2 linker distal from the spike-binding site. Importantly, the cell fusion-boosting function of HS can be targeted by an investigational HS-binding drug, which reduces syncytium formation in vitro and viral infection in mice. Thus, HS, as a host factor exploited by SARS-CoV-2 to facilitate receptor clustering and a stimulator of infection-associated syncytium formation, may be a promising therapeutic target for severe COVID., (© 2023. Springer Nature Limited.)

Background: Human respiratory syncytial virus (hRSV) is a key contributor to lower respiratory tract infections (LRTIs), affecting children aged 0-5 years and often leading to outpatient visits, emergency department utilization, and hospitalization. With the development of hRSV vaccines for mitigation, understanding the epidemiological impact of hRSV infections among 0-5-year-old pediatric outpatients in Italy is crucial., Methods: This systematic review conducted searches on PubMed, Embase, Scopus, and the International HTA Database, yielding 20,845 English and Italian records from January 2000 to July 2022., Results: Six eligible articles were identified following inclusion and exclusion criteria. These studies demonstrated hRSV-positivity proportions ranging from 18% to 41% in pediatric outpatients with respiratory infections. However, data comparability was hindered by diverse diagnostic approaches, data sources, sample populations, and study designs. Notably, hRSV-positivity showed temporal variability, rising from 23.8% (2001-2002) to 40.6% (2019-2020). This trend could stem from evolving epidemiological factors, heightened clinician awareness in hRSV diagnosis, or more sensitive molecular techniques., Conclusion: As the first review of its kind, this study underscores the need for more comprehensive data to inform effective preventive strategies against hRSV-related burdens in pediatric outpatients.

Apolipoprotein E (ApoE)'s ϵ4 alle is the most important genetic risk factor for late onset Alzheimer's Disease (AD). Cell-surface heparan sulfate (HS) is a cofactor for ApoE/LRP1 interaction and the prion-like spread of tau pathology between cells. 3-O-sulfo (3-O-S) modification of HS has been linked to AD through its interaction with tau, and enhanced levels of 3-O-sulfated HS and 3-O-sulfotransferases in the AD brain. In this study, we characterized ApoE/HS interactions in wildtype ApoE3, AD-linked ApoE4, and AD-protective ApoE2 and ApoE3-Christchurch. Glycan microarray and SPR assays revealed that all ApoE isoforms recognized 3-O-S. NMR titration localized ApoE/3-O-S binding to the vicinity of the canonical HS binding motif. In cells, the knockout of HS3ST1-a major 3-O sulfotransferase-reduced cell surface binding and uptake of ApoE. 3-O-S is thus recognized by both tau and ApoE, suggesting that the interplay between 3-O-sulfated HS, tau and ApoE isoforms may modulate AD risk., (© 2023 Wiley-VCH GmbH.)

The mechanism of syncytium formation, caused by spike-induced cell-cell fusion in severe COVID-19, is largely unclear. Here we combine chemical genetics with 4D confocal imaging to establish the cell surface heparan sulfate (HS) as a critical host factor exploited by SARS-CoV-2 to enhance spike’s fusogenic activity. HS binds spike to facilitate ACE2 clustering, generating synapse-like cell-cell contacts to promote fusion pore formation. ACE2 clustering, and thus, syncytium formation is significantly mitigated by chemical or genetic elimination of cell surface HS, while in a cell-free system consisting of purified HS, spike, and lipid-anchored ACE2, HS directly induces ACE2 clustering. Importantly, the interaction of HS with spike allosterically enables a conserved ACE2 linker in receptor clustering, which concentrates spike at the fusion site to overcome fusion-associated activity loss. This fusion-boosting mechanism can be effectively targeted by an investigational HS-binding drug, which reduces syncytium formation in vitro and viral infection in mice.

Heparanase, an endo-β-d-glucuronidase produced by a variety of cells and tissues, cleaves the glycosidic linkage between glucuronic acid (GlcA) and a 3-O- or 6-O-sulfated glucosamine, typified by the disaccharide -[GlcA-GlcNS3S6S]-, which is found within the antithrombin-binding domain of heparan sulfate or heparin. As such, all current forms of heparin are susceptible to degradation by heparanase with neutralization of anticoagulant properties. Here, we have designed a heparanase-resistant, ultralow molecular weight heparin as the structural analogue of fondaparinux that does not contain an internal GlcA residue but otherwise displays potent anticoagulant activity. This heparin oligosaccharide was synthesized following a chemoenzymatic scheme and displays nanomolar anti-FXa activity yet is resistant to heparanase digestion. Inhibition of thrombus formation was further demonstrated after subcutaneous administration of this compound in a murine model of venous thrombosis. Thrombus inhibition was comparable to that observed for enoxaparin with a similar effect on bleeding time.

Identifying determinants of COVID-19 vaccine uptake is essential for developing effective strategies for promoting vaccination. This longitudinal study aimed to explore predictors of actual COVID-19 vaccine uptake in workers involved in essential services during the first lockdown period in the Prato Province (Italy). All essential workers were invited and surveyed before COVID-19 vaccine approval (96.5% participation rate). Participants were followed up to evaluate their actual COVID-19 vaccination uptake using the vaccination register. Multinomial models were performed to assess predictors of delayed vaccination or non-vaccination. A total of 691 participants were included, of whom 21.7% had delayed the vaccination and 4.4% were unvaccinated. Participants with a sufficient level of health literacy were 50.2% in the vaccinated-on-time group and 32.3% in the unvaccinated group. The multinomial model predictors of delayed vaccination were work type (OR = 0.51), age between 50 and 59 years (OR = 1.82), and influenza vaccination uptake in the last season (OR = 2.51). Predictors of being unvaccinated were work type (OR = 0.33) and attitudes related to attributing less importance to COVID-19 preventive measures (OR = 0.47). Findings showed distinct predictors for COVID-19 vaccination delay and for being unvaccinated. Being unvaccinated seems to be associated with a general skepticism toward prevention measures.

This paper adds to the scant literature on the impact of terrorism on consumer behaviour, focusing on household spending on goods that are sensitive to brain-stress neurocircuitry. These include sweet- and fat-rich foods but also home necessities and female-personal-hygiene products, the only female-targeted good in our data. We examine unique continuous in-home-scanner expenditure data for a representative sample of about 15,000 French households, observed in the days before and after the terrorist attack at the Bataclan concert-hall. We find that the attack increased expenditure on sugar-rich food by over 5% but not that on salty food or soda drinks. Spending on home maintenance products went up by almost 9%. We detect an increase of 23.5% in expenditure on women's personal hygiene products. We conclude that these effects are short-lived and driven by the responses of households with children, youths, and those residing within a few-hours ride of the place of the attack., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Heparan sulfate (HS) and chondroitin sulfate (CS) are two structurally distinct natural polysaccharides. Here, we report the synthesis of a library of seven structurally homogeneous HS and CS chimeric dodecasaccharides (12-mers). The synthesis was accomplished using six HS biosynthetic enzymes and four CS biosynthetic enzymes. The chimeras contain a CS domain on the reducing end and a HS domain on the nonreducing end. The synthesized chimeras display anticoagulant activity as measured by both in vitro and ex vivo experiments. Furthermore, the anticoagulant activity of H/C 12-mer 5 is reversible by protamine, a U.S. Food and Drug Administration-approved polypeptide to neutralize anticoagulant drug heparin. Our findings demonstrate the synthesis of unnatural HS-CS chimeric oligosaccharides using natural biosynthetic enzymes, offering a new class of glycan molecules for biological research.

Heparan sulfate 3- O -sulfotransferase (3-OST) transfers a sulfo group to the 3-OH position of a glucosamine saccharide unit to form 3- O -sulfated heparan sulfate. 3- O -sulfation is known to be critically important for bestowing anticoagulant activity and other biological functions of heparan sulfate. Here, we report two ternary crystal structures of 3-OST-5 with PAP (3'-phosphoadenosine 5'-phosphate) and two octasaccharide substrates. We also used 3-OST-5 to synthesize six 3- O -sulfated 8-mers. Results from the structural analysis of the six 3- O -sulfated 8-mers revealed the substrate specificity of 3-OST-5. The enzyme prefers to sulfate a 6- O -sulfo glucosamine saccharide that is surrounded by glucuronic acid over a 6- O -sulfo glucosamine saccharide that is surrounded by 2- O -sulfated iduronic acid. 3-OST-5 modified 8-mers display a broad range of anti-factor Xa activity, depending on the structure of the 8-mer. We also discovered that the substrate specificity of 3-OST-5 is not governed solely by the side chains from amino acid residues in the active site. The conformational flexibility of the 2- O -sulfated iduronic acid in the saccharide substrates also contributes to the substrate specificity. These findings advance our understanding for how to control the biosynthesis of 3- O -sulfated heparan sulfate with desired biological activities., Competing Interests: Competing interest RW, AMK, ZW, ES, Jine Li, JMK and LCP declare no competing interest.

Heparin binds to and activates antithrombin (AT) through a specific pentasaccharide sequence, in which a trisaccharide subsite, containing glucuronic acid (GlcA), has been considered as the initiator in the recognition of the polysaccharide by the protein. Recently it was suggested that sulfated iduronic acid (IdoA2S) could replace this "canonical" GlcA. Indeed, a heparin octasaccharidic sequence obtained by chemoenzymatic synthesis, in which GlcA is replaced with IdoA2S, has been found to similarly bind to and activate antithrombin. By using saturation-transfer-difference (STD) NMR, NOEs, transferred NOEs (tr-NOEs) NMR and molecular dynamics, we show that, upon binding to AT, this IdoA2S unit develops comparable interactions with AT as GlcA. Interestingly, two IdoA2S units, both present in a 1 C 4 - 2 S 0 equilibrium in the unbound saccharide, shift to full 2 S 0 and full 1 C 4 upon binding to antithrombin, providing the best illustration of the critical role of iduronic acid conformational flexibility in biological systems., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A biological medicine (or biologicals) is a term for a medicinal compound that is derived from a living organism. By their very nature, they are complex and often heterogeneous in structure, composition and biological activity. Some of the oldest pharmaceutical products are biologicals, for example insulin and heparin. The former is now produced recombinantly, with technology being at a point where this can be considered a defined chemical entity. This is not the case for the latter, however. Heparin is a heterogeneous polysaccharide that is extracted from the intestinal mucosa of animals, primarily porcine, although there is also a significant market for non-porcine heparin due to social and economical reasons. In 2008 heparin was adulterated with another sulfated polysaccharide. Unfortunately this event was disastrous and resulted in a global public health emergency. This was the impetuous to apply modern analytical techniques, principally NMR spectroscopy, and multivariate analyses to monitor heparin. Initially, traditional unsupervised multivariate analysis (principal component analysis (PCA)) was applied to the problem. This was able to distinguish animal heparins from each other, and could also separate adulterated heparin from what was considered bona fide heparin. Taught multivariate analysis functions by training the analysis to look for specific patterns within the dataset of interest. If this approach was to be applied to heparin, or any other biological medicine, it would have to be taught to find every possible alien signal. The opposite approach would be more efficient; defining the complex heterogeneous material by a library of bona fide spectra and then filtering test samples with these spectra to reveal alien features that are not consistent with the reference library. This is the basis of an approach termed spectral filtering, which has been applied to 1D and 2D-NMR spectra, and has been very successful in extracting the spectral features of adulterants in heparin, as well as being able to differentiate supposedly biosimilar products. In essence, the filtered spectrum is determined by subtracting the covariance matrix of the library spectra from the covariance matrix of the library spectra plus the test spectrum. These approaches are universal and could be applied to biological medicines such as vaccine polysaccharides and monoclonal antibodies.

The heparan sulfate mimetic PI-88 is a complex mixture of sulfated oligosaccharides with anti-metastatic and anti-angiogenic activity due to its potent inhibition of heparanase and heparan sulfate-dependent angiogenic growth factors. It was recently in Phase III clinical trials for postresection hepatocellular carcinoma. The major oligosaccharide constituents of PI-88 were prepared for the first time by sulfonation of individually purified phosphorylated oligosaccharides isolated from the PI-88 precursor. PI-88 and its components were subjected to detailed 1D and 2D NMR spectroscopic analysis. The spectra of the individual components greatly assisted the assignment of minor resonances in the 1H NMR spectrum of PI-88. The data also showed that the majority of the oligosaccharides in PI-88 are fully sulfated and that undersulfated species present are largely due to anomeric desulfation. The solution conformation of the phosphomannopentaose sulfate (major component) of PI-88 was then determined by a combination of molecular dynamics simulations and NOE measurements which may provide insights into its binding interactions with target proteins.

Heparin is a highly sulfated glycosaminoglycan (GAG) of natural origin used as an anticoagulant and antithrombotic drug. These properties are principally based on the binding and activation of antithrombin (AT) through the pentasaccharide sequence GlcNAc/NS,6S-GlcA-GlcNS,3,6S-IdoA2S-GlcNS,6S (AGA*IA). Literature data show that the population of the 2 S 0 ring conformation of the 2-O-sulfo-α-l-iduronic acid (IdoA2S) motif correlates with the affinity and activation of AT. It was recently demonstrated that two synthetic AGA*IA-containing hexasaccharides (one G unit added at the reducing end), differing in the degree of sulfation of the IdoA unit, show comparable affinity and ability to activate AT, despite a different conformation of the IdoA residue. In this paper, the binding of these two glycans to AT was studied by isothermal titration microcalorimetry (ITC), transferred (tr-) NOESY, saturation transfer difference (STD) NMR spectroscopy and molecular dynamics (MD) simulations. Results indicated that both the IdoA2S and the IdoA units assume a 2 S 0 conformation when bound with AT, and so present a common binding epitope for the two glycans, centred on the AGA*IA sequence., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)