Campylobacter jejuni and Campylobacter coli are major foodborne pathogens. We announce the deposition of Sequential Window Acquisition of All Theoretical Mass Spectra (SWATH-MS) proteomic data sets from PBT2-treated and untreated cultures. These data sets provide a resource for investigating the proteomic response of Campylobacter spp. to the antimicrobial ionophore PBT2.

Accurate prediction of small-molecule bioactivity is critical for accelerating drug discovery. Here, we report MoActiPred, a multimodal AI framework that integrates 2D molecular graphs, physicochemical descriptors, and 3D E (3)-equivariant geometric representations. Using a curated ChEMBL-derived influenza virus neuraminidase (NA) inhibitor dataset (1245 compounds; IC50 threshold 20 nM), MoActiPred showed the strongest overall performance among the evaluated methods, including traditional machine-learning models (Random Forest, SVM, and XGBoost), SMILES-based language models (ChemBERTa, ChemFM, and PolyBERT), and graph-based baselines (MolCLR and SEGNN). On the public test set, MoActiPred obtained an accuracy of 0.952, MCC of 0.797, and F1 score of 0.898, while retaining high average precision, supporting the value of combining 2D and 3D representations. To evaluate its practical utility, MoActiPred was applied prospectively to 26 oseltamivir analogues designed, experimentally synthesised and evaluated against influenza A (H3N2) NA. The model correctly predicted 6 of 7 high-activity compounds (IC50 < 20 nM) and 18 of 19 inactive or weakly active derivatives, yielding 92.3

Abstract Thoeris defence systems protect bacteria from phages via abortive infection. In type I Thoeris systems, ThsA effectors containing silent information regulator 2 (SIR2) and SMF/DprA-LOG (SLOG) domains are activated by the cyclic ADP-ribose (ADPR) isomer 3′cADPR, triggering abortive infection via nicotinamide adenine dinucleotide (NAD + ) depletion. 3′cADPR activates the NADase activity of Bacillus subtilis ThsA tetramers via filament formation of its SIR2 domains, but the molecular details of how 3′cADPR triggers this process remain incompletely understood. Here, we demonstrate that ThsA activation by 3′cADPR-induced SIR2 filament formation is conserved in type I Thoeris systems from Streptococcus equi and Entercoccus facium. We present cryo-electron microscopy structures of the S. equ i ThsA filament bound to 3′cADPR and the non-cleavable NAD + analog carba-NAD + , and of the S. equi ThsA tetramer bound to 3′cADPR. These structures reveal that SIR2 filament formation is required to stabilise an active site conformation that can bind and hydrolyse NAD + . The structures also show that 3′cADPR induces quaternary alterations in the SLOG dimers and consequently the SIR2 tetramer to enable ThsA filament formation. Collectively, our study provides a comprehensive understanding of 3′cADPR-induced activation of type I Thoeris effectors.

COVID-19 disease is associated with thrombosis, but the pathogenic mechanism remains unclear. Here, we investigate how SARS-CoV-2 spike protein causes platelet activation and aggregation. Our three-dimensional ultrastructural analyses showed that invaginated platelet structures, open canalicular system (OCS), expanded upon activation, trapping viral particles in the process. Binding with platelet OCS concealed SAR-CoV-2 spike-coated particles from virion detection in platelet-depleted blood plasma. Both SARS-CoV-2 spike coated-particles and recombinant spikes specifically induced platelet aggregation with nanoscale filipodia extensions, with the terminal sialic acids of the SARS-CoV-2 spike protein-associated sialoglycoconjugates being the key determinant in platelet activation. Our work illustrates that virus-associated sialic acids, not proteins, are functionally responsible for SARS-CoV-2 induced thrombotic events, providing a mechanistic insight on how glycosylation contributes to disease severity in COVID-19. This study lays the foundation for the development of glycan-modified vaccines with reduced risks of thrombosis.

Background and objectives Enterococcus faecium and Staphylococcus aureus are opportunistic bacterial pathogens with a demonstrated capacity to develop antimicrobial resistance and cause serious life-threatening infections, underscoring the urgent need for new therapeutic options.Methods Here, we have synthesized and characterized the activities of an 8-hydroxyquinoline-based ionophore antibiotic (ionophoroantibiotic; IP antibiotic), designated 'IP-antibiotic 12.'Results Using multidrug-resistant strains of E. faecium and S. aureus, in vitro investigations revealed that IP-antibiotic 12 exhibits bactericidal activity, demonstrates a low propensity for resistance emergence, increases the susceptibility of particular strains to select antibiotics, possesses a favorable toxicity profile, and dysregulates bacterial metal homeostasis. IP-antibiotic 12 demonstrated therapeutic efficacy against multidrug-resistant S. aureus skin infection, as a direct-acting topical antimicrobial and antibiotic adjunct when co-administered with oral linezolid. Interestingly, it was not efficacious in murine models of systemic and pulmonary infection.Conclusions These results highlight the potential of IP-antibiotic 12 as a novel therapeutic against multidrug-resistant gram-positive bacteria and provide a foundation for the development of next-generation IP-antibiotics with enhanced in vivo therapeutic efficacy.

Human noroviruses are a leading cause of acute gastroenteritis, yet no licensed vaccine or antiviral is currently available. The extensive genetic diversity of noroviruses and the limited cross-protective immunity between genotypes complicate the development of broadly effective interventions. To systematically characterize norovirus-specific B cell responses from healthy individuals, we applied linking B cell receptor to antigen specificity through sequencing (LIBRA-seq) to peripheral blood mononuclear cells using a genetically diverse panel of norovirus capsid protruding (P) domain antigens. Antigen-specific B cells were identified at single-cell resolution and selected for recombinant monoclonal antibody (mAb) expression. A total of 25 norovirus-specific mAbs were generated and evaluated for binding breadth across multiple genogroup II (GII) genotypes. Binding analyses revealed substantial heterogeneity, with some mAbs displaying narrow, genotype-restricted recognition, whereas others exhibited broad cross-reactivity. Epitope competition assays using a subset of mAbs demonstrated both overlapping and distinct binding sites on the P domain. Functional activity, assessed using a surrogate neutralization assay measuring inhibition of histo-blood group antigen (HBGA) binding, showed that several broadly reactive antibodies inhibited virus-like particle attachment to HBGAs with variable potency across genotypes, whereas others exhibited genotype-restricted inhibition. Together, these results demonstrate that LIBRA-seq enables efficient mapping of human antibody repertoires to norovirus at the population level and reveals that natural exposure generates a diverse B-cell repertoire with varying specificity and functional breadth.IMPORTANCEHuman noroviruses are the leading cause of acute viral gastroenteritis worldwide, yet individuals experience repeated infections throughout life, indicating that protective immunity is incomplete and remains poorly understood. In this study, we applied the high-throughput LIBRA-seq approach to systematically map the norovirus-specific antibody repertoire in healthy human donors, enabling the identification of monoclonal antibodies that recognize and functionally inhibit clinically relevant genotypes. This work provides new insight into the breadth and specificity of naturally acquired humoral immunity by establishing a comprehensive framework for analyzing antigen-specific B cell responses in the general population and generates a well-defined panel of human antibodies that will be useful for future studies of norovirus immunity, vaccine evaluation, and therapeutic development.

Helicobacter pylori colonizes the gastric mucosa of around half of the world's population and is a major cause of chronic gastritis, peptic ulcer disease, and gastric cancer. Current therapies are becoming increasingly ineffective due to the rapid spread of antibiotic resistance, creating an urgent need for new treatment options with distinct mechanisms of action. Drug repurposing offers a practical and cost-effective approach to address this gap. PBT2 is an 8-hydroxyquinoline derivative originally developed for the treatment of neurodegenerative diseases and has more recently been shown to possess antimicrobial activity. In this study, we demonstrate that PBT2 displays potent bactericidal activity against H. pylori, including multidrug-resistant clinical isolates. PBT2 rapidly killed H. pylori in vitro at low concentrations, with faster killing kinetics than commonly used antibiotics, and no resistance was detected after 30 days of continuous exposure. Importantly, PBT2 was effective in clearing an H. pylori infection in a murine model. Quantitative sequential window acquisition of all theoretical-mass spectrometry proteomic analysis revealed that PBT2 triggers broad disruption of essential bacterial processes, including global suppression of translation, impairment of iron-sulfur cluster assembly and respiration, dysregulation of metal homeostasis, and reduced abundance of virulence- and motility-associated proteins. We reported that PBT2 can act as a nickel ionophore, with Ni2+ being the highest-affinity ligand for PBT2 reported to date. Together, these findings suggest that PBT2 acts through a multifaceted, metal-dependent mode of action that limits the potential for emergence of resistance. Our work highlights PBT2 as a promising candidate for repurposing to treat multidrug-resistant H. pylori infections.IMPORTANCEAntibiotic resistance is steadily reducing our ability to treat common bacterial infections, while the development of new antibiotics has slowed. Helicobacter pylori is a clear example of this growing problem, with treatment failures becoming more common worldwide. This study highlights the value of taking a different approach by repurposing existing drugs for new antibacterial uses. Rather than acting on a single bacterial target, the compound examined here disrupts multiple essential processes at once, reducing the probability of resistance developing.

Background Intrahepatic cholangiocarcinoma (ICC) is a highly malignant tumor with limited therapeutic options. Galectin−1 (Gal−1) has been implicated in tumor progression in several cancers; however, its roles in ICC remain unclear. This study examined the role of Gal−1 in ICC progression and characterized its molecular mechanisms. Methods The expression of Gal−1 in ICC was evaluated by Western blot analysis and immunohistochemistry, and its functional effects on ICC cell proliferation and migration were assessed through Western blot analysis, wound healing assays, CCK8 assays, and in vivo knockdown models. Transcriptome sequencing was performed on Gal-1-deficient cells to identify downstream effectors. Fructose−1,6−bisphosphatase 1 (FBP1) was identified as a key candidate and further validated in clinical samples and functional assays. The involvement of the RAS/ERK signaling pathway was investigated, and mechanistic causality was confirmed through FBP1 overexpression and rescue experiments. Results Gal−1 was significantly overexpressed in ICC and correlated with poor prognosis. Gal−1 knockdown markedly inhibited ICC cell proliferation and migration both in vitro and in vivo. Mechanistically, suppression of Gal−1 resulted in upregulation of FBP1 and inhibition of the RAS/ERK signaling pathway. Conclusion Our findings identify a previously unrecognized Gal−1/FBP1/RAS-ERK regulatory axis driving ICC progression, highlighting Gal−1 as a possible therapeutic target and prognosis indicator. However, further investigation is required to elucidate Gal−1 detailed regulatory mechanisms in ICC.

Sialic acid O-acetylation is implicated in the modulation of sialoglycan recognition and ganglioside biology. The sugar modification is catalyzed by CASD1, a Golgi membrane protein that encompasses a luminal catalytic domain and a multipass transmembrane domain. The mechanism of how acetyl-CoA is provided to the Golgi remains poorly understood. Here, we show that the acetyl-CoA transporter SLC33A1 provides acetyl-CoA to the luminal domain of CASD1 and that patient-derived SLC33A1 variants linked to inherited neurodevelopmental and neurodegenerative disorders impair ganglioside 9-O-acetylation. Under conditions that enable the formation of 7,9-di-O-acetylated sialoglycans, genetic inactivation of SLC33A1 impaired di-O-acetylation, but unexpectedly, still enabled mono-O-acetylation. Structure prediction and site-directed mutagenesis revealed a second active site in CASD1 that shares striking similarities with the catalytic acetyl-CoA binding transmembrane tunnel of the lysosomal acetyltransferase HGSNAT. Together, our data provide strong evidence that CASD1 has dual functionalities and catalyzes 7,9-di-O-acetylation through SLC33A1-dependent luminal acetylation and SLC33A1-independent transmembrane acetylation.

Cell surface sialylation is utilized by a number of pathogenic bacteria to evade the host immune system through molecular mimicry of host sialoglycoconjugates. Human pathogen Neisseria meningitidis serotype B (NmB) expresses both sialylated capsule and surface lipooligosaccharides as pivotal virulence factors. An essential enzyme in the sialylation pathway of NmB is CMP-sialic acid synthetase (CSS), which produces the activated nucleotide sugar necessary for sialic acid transfer. In this work, novel C-4, -5, -7, and -9 functionalized derivatives of neuraminic acid β-methyl glycoside (Neuβ2Me) were synthesized as candidate CSS inhibitors. A number of these were found to reduce the activity of NmB CSS in vitro. The highest inhibition of NmB CSS, in a mixed mode manner, was observed with a Neu5Acβ2Me C-9 serine carboxamide. Direct interaction with the enzyme was confirmed by Saturation Transfer Difference (STD) NMR. Supplementation of growth media with this compound reduced lipooligosaccharide (LOS) sialylation of living N. meningitidis, thus providing an interesting starting point for the development of specific NmB CSS inhibitors as an alternative treatment strategy to fight bacterial infections.

Human parainfluenza virus 3 is a highly abundant RNA virus that primarily affects young children, the elderly, and immunocompromised individuals, leading to severe lower respiratory infections and pneumonia. Despite an urgent need of treatment options for these high-risk patients, neither a vaccine nor specific antiviral are currently approved. Blocking viral entry by targeting the viral surface glycoprotein haemagglutinin-neuraminidase (HN) has shown promising results in vitro and, to some extent, in vivo. However, to further evaluate these antiviral approaches for potential human application, a detailed understanding of early hPIV-3 infection and drug treatment mechanisms in human lung tissue is needed. In this study, we established a model for early hPIV-3 infection in human precision-cut lung slices (PCLS). We demonstrate specific infection of small airway epithelial cells followed by a distinct antiviral and inflammatory response marked by expression and secretion of type I, II and III interferons, chemokines such as IP-10 and ITAC, and pro-inflammatory markers IL-6 and TNF-α, but only limited induction of cytokines associated with high clinical severity, such as IL-8. Prophylactic treatment with two viral entry HN-inhibitors significantly reduced hPIV-3 viral load and inflammatory response after infection in the human lung tissue slices, demonstrating the high usability of the PCLS infection model for pharmacological assessment of novel antiviral drugs.

Human parainfluenza virus type 3 (HPIV-3) remains a major cause of respiratory illness particularly among young children, the elderly and immunocompromised individuals. Despite significant efforts in therapeutic discovery research, there is neither an effective antiviral nor a vaccine available against HPIV-3. Host cell glycosylation is known to play a pivotal role in virus entry and replication. While some host glycan-based cellular receptors for HPIV-3 have been identified, the dynamics of the host-cell glycome upon HPIV-3 infection has never been studied. Herein, we report the first mass spectrometry-based study that provides direct insight into the remodelling of the human lung adenocarcinoma cell (A549) glycome upon HPIV-3 infection. Our results reveal significant remodelling of host-cell glycome, including increased expression of oligomannose and reduced expression of sialylated complex-type N -glycans. In addition, we observed altered O -glycan expression, with upregulation of core 1 and downregulation of core 2 type structures in infected cells compared to mock-infected controls. HPIV-3 infection also led to distinct remodelling of glycosphingolipid glycans. Together, these findings present the first evidence that HPIV-3 infection alters host-cell glycome, offering new insights into the virus’ impact on host-cellular processes. ### Competing Interest Statement The authors have declared no competing interest. * HPIV : Human parainfluenza virus HN : haemagglutinin-neuraminidase H.p.i : Hour’s post-infection CEP : Cell extracted protein SP : Secreted protein HN : N -Acetyl Glucosamine H : Hexose dH : Deoxyhexose Man : Mannose N : N -Acetyl Neuraminic Acid G : N -glycolylneuraminic Acid GSL : Glycosphingolipids PGC : Porous graphitized carbon ESI : Electrospray ionization CID : Collision induced dissociation National Health and Medical Research Council, https://ror.org/011kf5r70, GNT1196520, GNT1157150, APP1047824

3-Fluoroneuraminosyl fluorides are invaluable probes for studying the catalytic mechanism of sialidases (neuraminidases), and as sialidase inhibitors. Significantly, when a C-3 equatorial fluorine is installed on a C-4 functionalised N-acylneuraminic acid (Neu)-based template, the compounds are potent and selective inhibitors of both influenza and parainfluenza sialidases, and of virus replication. Typically, the reported syntheses of 3-fluoroneuraminosyl fluorides involve either an enzymatic or a chemical synthesis that have uncontrolled stereoselectivity in the introduction of fluorine at C-3 of Neu and consequently yield a mixture of C-3 ax and C-3 eq fluoro derivatives. We now report a simple approach for the exquisitely stereo-controlled introduction of the C-3 equatorial fluorine on Neu by incorporation of steric bulk at C-4. Through this method, we have elaborated a novel synthetic route that exclusively produces the potent anti-influenza drug candidate; 2,3-difluoro-zanamivir analogue with C-3 eq fluoride.

Nipah (NiV) and Hendra viruses (HeV) have emerged as deadly zoonotic pathogens over the last three decades. Like all paramyxoviruses, Henipaviruses utilize a surface glycoprotein to attach to and invade targeted cells. Inhibiting this attachment glycoprotein is a promising strategy for developing effective antihenipaviral drugs. A multidisciplinary approach has been employed to investigate the structures of HeV and NiV attachment glycoproteins, identifying a flexible region near their binding site. This region, loop 240, can adopt an open conformation in unliganded attachment glycoproteins and a closed "latch" conformation in the presence of their cognate receptor Ephrin B2. Site-directed mutagenesis of the HeV attachment glycoproteins has shown that the engagement of R242 with Ephrin B2 plays an important role in the binding mechanism. This discovery provides greater insight into the dynamic nature of henipaviral attachment proteins and has implications for antiviral drug development.

Influenza viruses are a major cause of respiratory illness, with significant public health impact due to their ability to cause pandemics. This dialogue brought together leading experts including Professors George Fu Gao, Stephen Cusack, Mark von Itzstein, Ervin Fodor, Jonathan Grimes, Aartjan J.W. te Velthuis, and Tao Deng to decode the pressing scientific challenges and future directions in influenza research. They discussed how structural studies of the influenza polymerase have advanced our understanding of viral RNA transcription and replication. These insights are crucial for developing new antiviral drugs, with a particular focus on targeting the polymerase and its interactions with host factors like acidic nuclear phosphoprotein 32 (ANP32). The dialogue also highlighted the potential of AI to assist in designing small molecule drugs, offering new strategies for combating influenza. Future research will continue to unravel the complexities of the polymerase’s role in replication, aiming to translate these findings into effective therapies and resilient public health strategies.

A large‐scale chemoenzymatic methodology has been developed to prepare a natural and substituted analogues of the Glycophorin A tetrasaccharide, in which the α‐2,3‐sialic acid is modified at either C5 or C9 either as F, OMe, CH 3 , glycolyl or N 3 . The strategy involved a chemical synthesis of a Gal‐β‐1,3‐GalNAc disaccharide in which the galactose remains acetylated to first enable selective α‐2,6‐sialylation using a one‐pot aldolase‐NmCSS‐Pd2,6‐ST enzyme system. Finally, C5 or C9 modifed α‐2,3 sialosides were placed at the galactoside moiety using C2 or C6 modified MaNAc derivatives catalyzed by aldolase NmCSS‐Pd2,3‐ST enzyme combination. The synthesised molecules can be useful in binding studies with a protein of interest or as an entry point to establish a library of analytical samples for mass‐spectrometric studies.