For many decades, there have been numerous reported cases of falsified liquid medical products, including vaccine and insulin preparations worldwide, but to date, there has been a lack of affordable and accessible analytical methods for biological medicines and vaccine authenticity testing. A conventional clinical chemistry analyser (Abbott Architect c16000) was used to determine the concentrations of analytes in genuine liquid biological products (vaccines and insulin) and falsified vaccine surrogates. Eight analytes were measured for each sample: sodium, potassium, chloride, calcium, magnesium, phosphate, glucose and protein. Each genuine liquid product had unique concentrations of analytes when tested using the eight methods applied, allowing clear differentiation from the falsified surrogates. In a blinded study, reproducibility was significantly high when the samples were run intra- and inter-batch up to 9 times over 9 different days, and it was possible to identify most of the samples by analyte presence alone. Imprecision was < 1.0 CV% for ion-selective electrode methods and typically < 5 CV% for spectrophotometric methods. A decision tree was created which was able to identify all samples. We demonstrate for the first time that a conventional clinical chemistry analyser provides a low-cost method to accurately differentiate genuine products from falsified surrogate liquid medicines and vaccines. This novel method has the potential to be used globally due to widespread use of clinical chemistry analysers in hospitals across the world, including in low- and middle-income countries where many cases of falsified medicines have been identified.

Ovarian cancer remains a lethal malignancy due to chemoresistance and toxicity, which limits the dose of chemotherapy that can be used, necessitating the development of more targeted therapies such as antibody-drug conjugates (ADCs). However, conventional ADCs suffer from heterogeneity. This study aimed to develop a stable, homogeneous ADC by utilizing a bifunctional dibromomaleimide (DBM) linker to cross-link antibody cysteine residues via disulfide-bridging. A DBM linker was synthesized from 3,4-dibromofuran-2,5-dione and bound to the cytotoxic agent triptolide. This triptolide payload was then conjugated to trastuzumab via site-specific disulfide rebridging to yield a homogeneous trastuzumab-triptolide conjugate to target human epithelial growth factor receptor 2 (HER2) on ovarian cancer cells. The study evaluated the ADC's efficacy against SKOV-3 (high HER2 expression) and OVCAR-8 (low HER2 expression) cell lines. The results showed that the ADC was slightly more potent in SKOV-3 cells, yielding lower IC50 values compared to OVCAR-8. Mechanistic studies of the ADC via flow cytometry revealed that the ADC induced significant apoptosis and cell cycle arrest, characterized by a concentration-dependent increase in Caspase-3/7 expression and distinct alterations in cell population distribution. Furthermore, ADC treatment led to a concentration-dependent decrease in HER2 levels in SKOV-3 cells, confirming successful targeting. The study demonstrates that converting conventional maleimides into bifunctional DBM linkers allows the production of a homogeneous ADC via disulfide-bridging. This approach offers a promising strategy for developing potent anticancer therapeutics with improved selectivity for HER2-overexpressing ovarian cancers.

Delivery and utilisation of oxygen are critical determinants of skeletal muscle function, and therefore aerobic performance. Angiogenesis, the process of microvascular bed expansion, may be initiated by several tissue-level stimuli (e.g. of haemodynamic, myogenic or metabolic origin), which are typically present during dynamic exercise. Understanding the relative contribution of these distinct physiological stimuli to skeletal muscle remodelling is needed to develop effective therapeutic strategies to alleviate impaired tissue oxygen supply. In the present study, we uncoupled the predominantly mechanotransductive (i.e. elevated vascular shear stress and cyclical muscle activation) and predominantly chemotransductive (i.e. local tissue hypoxia) stimuli present during exercise by exposing C57b6 mice to either indirect muscle stimulation (10 Hz; ST) or systemic hypoxia (10% oxygen; H), for 7 days, respectively. Furthermore, we combined these stimuli (H+ST) to determine whether the effects were additive. After 7 days of intervention, the tibialis anterior muscle was sampled for histological quantification of microvascular supply and metabolomics analysis. We showed that ST promoted a significant angiogenic response within the muscle whereas H did not. Interestingly, the combined H+ST group had a blunted angiogenic response. Branch-chain amino acid levels were significantly decreased following ST, H and H+ST, consistent with an increased metabolic requirement for ATP, which represents an energy deficit. Proximate metabolites of the glycolytic pathway were significantly reduced following hypoxia, but not stimulation. Together, these observations are commensurate with mechanotransduction triggering structural remodelling of muscle that preserves the metabolome of muscle tissue, whereas chemotransduction inhibits the angiogenic response induced by ST, possibly as a consequence of altered glycolytic metabolism. KEY POINTS: Angiogenesis, the process of microvascular bed expansion, may be initiated by several tissue-level stimuli (e.g. haemodynamic, myogenic or metabolic in origin), which are typically present during dynamic exercise. There has been controversy about the structural (capillary) response of skeletal muscle to altered O2 status, involving decreased supply (hypoxia) or increased demand (activity). Here, we demonstrate that 7 days of activation of skeletal muscle by indirect electrical stimulation led to significant expansion of the capillary bed. However, a similar adaptive structural response was not observed following hypoxia. When combining indirect stimulation and hypoxia, hypoxia appears to blunt structural remodelling. Proximate metabolites of the glycolytic pathway were significantly reduced following hypoxia, but not stimulation. Together, these observations suggest that mechanotransduction (via indirect stimulation) triggers structural remodelling of muscle that preserves the metabolome of muscle tissue, whereas chemotransduction (via hypoxia) inhibits the angiogenic response induced by stimulation, possibly because of altered glycolytic metabolism.

Background: Mild cognitive impairment (MCI) is a heterogeneous state between normal ageing and dementia, often considered prodromal to Alzheimer's disease (AD). Progression is variable, and distinguishing stable from progressive MCI remains difficult, particularly in the presence of mixed neuropathology. Blood biomarkers such as phosphorylated tau181 (pTau181), glial fibrillary acidic protein (GFAP), and neurofilament light chain (NfL) demonstrate prognostic value in established AD, but limited performance for prognosticating progression from MCI. Methods: Blood protein biomarkers (pTau181, GFAP, NfL) were integrated with NMR- and LC-MS-derived metabolomic features. In a deeply phenotyped MCI cohort (VITACOG; n=68) with two-year MRI follow-up, cross-validated logistic regression identified discriminative multi-analyte panels to distinguish stable from progressive MCI. Disease progression was defined by worsening cortical atrophy, measured via annualised brain volume loss. Generalisability was tested in a larger community-based cohort from UK Biobank (n=223) and two Oxford Project to Investigate Memory and Ageing (OPTIMA) subsets with histopathological diagnosis (n=61, n=37). Results: Integration of pTau181 with six metabolite features yielded the highest prognostic performance (AUC 0.91; accuracy 80%), with metabolomic findings independently validated in the OPTIMA cohort. A complementary GFAP-NMR panel also performed strongly (AUC 0.80; accuracy 75%). In contrast, individual metabolites, including the atrophy marker homocysteine, and standalone protein biomarkers performed poorly (AUC ≤0.66), as well their combination (AUC 0.68), highlighting the added value of multi-omic integration. In an asymptomatic ageing population (UK Biobank), the models served as a population-level stress test, confirming that multi-omic integration improved specificity for MRI-derived atrophy measures and captured atrophy-related risk in community cohorts. Conclusion: Multi-omic integration of protein and metabolic features markedly improved prognostication of MCI progression by capturing early neurodegenerative signatures, yielding translational panels suitable for scalable risk stratification and early therapeutic intervention in clinical practice. ### Competing Interest Statement A.D.S. and H.R. are named as inventors on two patents held by the University of Oxford on the use of B-vitamins to treat cognitive disorders (US9364497 and US10966947). F.J. is named as an inventor on US10966947. These patents have been licensed to Elysium Health, NY. J.S.O.M. has a research contract and equipment loan from ThermoFisher Scientific, which manufactures IC-MS systems. S. de J., Q. G. and E. S. are employees of Numares AG (Am Biopark 9, 93053 Regensburg-Grass, Germany). All other authors report no conflicts of interest. N.J.A. received consultancy or speaker fees from BioArtic, Biogen, Lilly, Quanterix and Alamar Biosciences. H.Z. has served at scientific advisory boards and/or as a consultant for Abbvie, Acumen, Alector, Alzinova, ALZpath, Amylyx, Annexon, Apellis, Artery Therapeutics, AZTherapies, Cognito Therapeutics, CogRx, Denali, Eisai, Enigma, LabCorp, Merck Sharp & Dohme, Merry Life, Nervgen, Novo Nordisk, Optoceutics, Passage Bio, Pinteon Therapeutics, Prothena, Quanterix, Red Abbey Labs, reMYND, Roche, Samumed, ScandiBio Therapeutics AB, Siemens Healthineers, Triplet Therapeutics, and Wave, has given lectures sponsored by Alzecure, BioArctic, Biogen, Cellectricon, Fujirebio, LabCorp, Lilly, Novo Nordisk, Oy Medix Biochemica AB, Roche, and WebMD, is a co-founder of Brain Biomarker Solutions in Gothenburg AB (BBS), which is a part of the GU Ventures Incubator Program, and is a shareholder of CERimmune Therapeutics (outside submitted work). S. M. S. is co-founder and part-owner of SBGneuro. ### Funding Statement T.K. is funded by an EPSRC Postdoctoral Pathway Scheme (EP/Z534870/1) and EPSRC talent and skills funding and Numares AG (Am Biopark 9, 93053 Regensburg-Grass, Germany). This research was supported in part by the Aqua-Synapse EU framework (2022-2026) to D.A., funded by the European Union's Horizon 2020 Research and Innovation programme under the Marie Sklodowska-Curie Grant Agreement No. 101086453. The authors are solely responsible for the content of this publication, which does not necessarily represent the official views of the European Union or the European Research Executive Agency. H.Z. is a Wallenberg Scholar and a Distinguished Professor at the Swedish Research Council supported by grants from the Swedish Research Council (#2023-00356, #2022-01018 and #2019-02397), the European Union's Horizon Europe research and innovation programme under grant agreement No 101053962, and Swedish State Support for Clinical Research (#ALFGBG-71320). The original VITACOG trial was supported by grants from Charles Wolfson Charitable Trust, Medical Research Council, Alzheimer's Research Trust, Henry Smith Charity, John Coates Charitable Trust, Thames Valley Dementias and Neurodegenerative Diseases Research Network of the National Institute for Health Research, UK, and the Sidney and Elizabeth Corob Charitable Trust. The original OPTIMA cohort was supported by grants from Bristol-Myers Squibb, Medical Research Council and the Charles Wolfson Charitable Trust. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The VITACOG trial was approved by a local NHS Research Ethics Committee (COREC 04/Q1604/100). For the OPTIMA cohort, ethical approval was obtained from the Frenchay Research Ethics Committee (REC Ref 09/H0107/9). All participants provided written informed consent in accordance with the Declaration of Helsinki. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes Anonymised data not published within this article will be made available by request from any qualified investigator.

The CpG island methylator phenotype (CIMP) occurs in many colorectal cancers (CRCs). CIMP is closely associated with global hypermethylation and tends to occur in proximal tumours with microsatellite instability (MSI), but its origins have been obscure. A few CRCs carry oncogenic (gain-of-function) mutations in isocitrate dehydrogenase IDH1 . Whilst IDH1 is an established CRC driver gene, the low frequency of IDH1 -mutant CRCs (about 0.5%) has meant that the effects and molecular covariates of those mutations have not been established. We first showed computationally that IDH2 is also a CRC driver. Using multiple public and in-house CRC datasets, we then identified IDH mutations at the hotspots ( IDH1 codons 132 and IDH2 codons 140 and 172) frequently mutated in other tumour types. Somatic IDH mutations were associated with BRAF mutations and expression of mucinous/goblet cell markers, but not with KRAS mutations or MSI. All IDH-mutant CRCs were CIMP-positive, mostly at a high level. Cell and mouse models showed that IDH mutation was plausibly causal for DNA hypermethylation. Whilst the aetiology of hypermethylation generally remains unexplained, IDH-mutant tumours did not form a discrete methylation subcluster, suggesting that different underlying mechanisms can converge on similar final methylation phenotypes. Although further analysis is required, IDH mutations may be the first cause of hypermethylation to be identified in a common cancer type, providing evidence that CIMP and DNA methylation represent more than aging-related epiphenomena. Cautious exploration of mutant IDH inhibitors and DNA demethylating agents is suggested in managing IDH-mutant CRCs. © 2025 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Background PDE6H encodes PDE6γ′, the inhibitory subunit of the cGMP-specific phosphodiesterase 6 in cone photoreceptors. Inhibition of PDE6, which has been widely studied for its role in light transduction, increases cGMP levels. The purpose of this study is to characterise the role of PDE6H in cancer cell growth. Methods From an siRNA screen for 487 genes involved in metabolism, PDE6H was identified as a controller of cell cycle progression in HCT116 cells. Role of PDE6H in cancer cell growth and metabolism was studied through the effects of its depletion on levels of cell cycle controllers, mTOR effectors, metabolite levels, and metabolic energy assays. Effect of PDE6H deletion on tumour growth was also studied in a xenograft model. Results PDE6H knockout resulted in an increase of intracellular cGMP levels, as well as changes to the levels of nucleotides and key energy metabolism intermediates. PDE6H knockdown induced G1 cell cycle arrest and cell death and reduced mTORC1 signalling in cancer cell lines. Both knockdown and knockout of PDE6H resulted in the suppression of mitochondrial function. HCT116 xenografts revealed that PDE6H deletion, as well as treatment with the PDE5/6 inhibitor sildenafil, slowed down tumour growth and improved survival, while sildenafil treatment did not have an additive effect on slowing the growth of PDE6γ′-deficient tumours. Conclusions Our results indicate that the changes in cGMP and purine pools, as well as mitochondrial function which is observed upon PDE6γ′ depletion, are independent of the PKG pathway. We show that in HCT116, PDE6H deletion replicates many effects of the dark retina response and identify PDE6H as a new target in preventing cancer cell proliferation and tumour growth.

There have been hundreds of child deaths due to contamination of medicinal syrups with diethylene glycol (DEG) and ethylene glycol (EG). Detection of DEG and EG is usually performed by gas chromatography, a method that is costly, laborious, time-consuming, and not readily available in many low- and middle-income countries (LMICs). Thin-layer chromatography is relatively lower cost and portable; however, as with gas chromatography, it requires time and trained personnel. Alternative rapid, low-cost and simple methods to determine DEG/EG contamination are needed. We tested the suitability of enzymatic, chemical and antibody-based assays to determine DEG/EG. Assays using alcohol dehydrogenase and aldehyde dehydrogenase alone as well as in combination with glycolate oxidase could determine EG in raw materials and at less than 0.1% m/m in some finished products. Saliva and breast milk alcohol test strips, containing alcohol oxidase and costing $1, could determine EG with a detection limit of 0.5 to 2% m/m in under 2 minutes. Disposable breathalysers, costing only $1, could determine both DEG and EG from other alcohols in only 10 seconds. The methods described provide simple, rapid and low-cost alternatives to help determine DEG and EG contamination in pharmaceutical supply chains to help prevent deaths where equipment and trained human resources are limited.

We conducted PET imaging with [18F]FDOPA and dopamine D2/3 receptor ligand [18F]fallypride in aged transgenic rats carrying human pathogenic LRRK2 R1441C or G2019S mutations. These rats have mild age-dependent deficits in dopamine release restricted to dorsal striatum despite no overt loss of dopamine neurons or dopamine content and demonstrate L-DOPA-responsive movement deficits. LRRK2 mutant rats displayed no deficit in [18F]FDOPA uptake, consistent with intact dopamine synthesis in striatal axons. However, LRRK2-R1441C rats demonstrated greater binding of [18F]fallypride than LRRK2-G2019S or non-transgenic controls, from a regionally selective increase in dorsal striatum. Immunocytochemical labelling post-mortem confirmed a greater density of D2 receptors in LRRK2-R1441C than other genotypes restricted to dorsal striatum, consistent with upregulation of D2-receptors as a compensatory response to the greater dopamine release deficit previously demonstrated in this genotype. These results show that [18F]fallypride PET imaging is sensitive to dysregulation of dopamine signalling in the LRRK2-R1441C rat, revealing upregulation of D2 receptors that parallels observations in human putamen in early sporadic PD. Future studies of candidate therapies could exploit this non-invasive approach to assess treatment efficacy.

Maintaining cold-chain integrity is vital for vaccines to ensure that they remain within the recommended temperature limits during routine storage and transportation. This ensures vaccine stability, efficacy, and avoids degradation. Here, we propose rapid and low-cost tests based on simple glucose assays to detect heat-exposed degraded sucrose-containing vaccines through sucrose's inherent gradual conversion to glucose when exposed to elevated temperatures. Bioluminescent and colorimetric assays and a clinical biochemical analyser for urine samples could successfully determine effects of heat exposure on vaccines by detecting a significant increase in glucose levels. We show that this increase in glucose also correlates with the loss of vaccine potency. When vaccines were incubated at 37 and 45 °C, the bioluminescent assay was able to detect an increase in glucose levels from 12 h of heat exposure. The biochemical analyser could successfully detect increased glucose levels in a COVID-19 vaccine which had been exposed to 37 and 45 °C. Most importantly, the colorimetric assay has the advantage of producing a colour change visually upon simply mixing the vaccine with a reagent without the need for a plate reader or any other sophisticated devices. To our knowledge, this is the first simple, rapid and device-free test of its kind to detect heat-exposed substandard vaccines, making it a potential test for deploying at key points in the supply chain in warm and hot countries to check the integrity of vaccine cold-chain. Although this test does not replace the more definitive lot release assays such as potency assays, it could initially be used as a rapid and low-cost test to identify substandard sucrose-containing vaccines within supply chains, in support of WHO's Prevent, Detect, and Respond strategy.

We investigate the potential of Spatially Offset Raman Spectroscopy (SORS) as a rapid, non-invasive screening tool deployable in the field to detect diethylene glycol (DEG) and ethylene glycol (EG) in medicinal syrups within closed containers. Measurements were performed on neat propylene glycol (PG) and glycerol, key components of many medicinal syrups, as well as marketed medicinal syrup formulations spiked with DEG and EG at various concentration levels to assess the technique's limit of detection in real-life samples. SORS was able to detect these down to ∼0.5 % concentration level in neat PG for both DEG and EG compounds and ∼1 % concentration level for DEG and EG in neat glycerol. The DEG and EG detection thresholds for the marketed formulations measured through original bottles was ∼1 %, for Benylin (active ingredient: Glycerol) and Piriteze (active ingredient: Cetirizine Hydrochloride). For Calpol (active ingredient: Paracetamol) the detection limit was higher, ∼2 % for EG and ∼5 % for DEG. Although not reaching the International Pharmacopeial 0.1 % detection threshold currently required for purity checks for human consumption, the method can still be used to detect products where DEG or EG has been wrongly used instead of PG or glycerol or if present in large quantities. The technique could also be used for raw material identification testing to ensure no mislabelling has occurred in pre-production stages and as a screening device in distribution chains to detect major deviations from permitted content in non-diffusely scattering, clear formulations, to help prevent serious adverse outcomes, such as acute renal failure and deaths.

The rapid development and worldwide distribution of COVID-19 vaccines is a remarkable achievement of biomedical research and logistical implementation. However, these developments are associated with the risk of a surge of substandard and falsified (SF) vaccines, as illustrated by the 184 incidents with SF and diverted COVID-19 vaccines which have been reported during the pandemic in 48 countries, with a paucity of methods for their detection in supply chains. In this context, matrix-assisted laser desorption ionisation-time of flight (MALDI-ToF) mass spectrometry (MS) is globally available for fast and accurate analysis of bacteria in patient samples, offering a potentially accessible solution to identify SF vaccines. We analysed the COVISHIELD™ COVID-19 vaccine; falsified versions of which were found in India, Myanmar and Uganda. We demonstrate for the first time that analysis of spectra from the vaccine vial label and its adhesive could be used as a novel approach to detect falsified vaccines. Vials tested by this approach could be retained in the supply chain since it is non-invasive. We also assessed whether MALDI-ToF MS could be used to distinguish the COVISHIELD™ vaccine from surrogates of falsified vaccines and the effect of temperature on vaccine stability. Both polysorbate 80 and L-histidine excipients of the genuine vaccine could be detected by the presence of a unique combination of MALDI-ToF MS peaks which allowed us to distinguish between the genuine vaccines and falsified vaccine surrogates. Furthermore, even if a falsified product contained polysorbate 80 at the same concentration as used in the genuine vaccine, the characteristic spectral profile of polysorbate 80 used in genuine products is a reliable internal marker for vaccine authenticity. Our findings demonstrate that MALDI-ToF MS analysis of extracts from vial labels and the vaccine excipients themselves can be used independently to detect falsified vaccines. This approach has the potential to be integrated into the national regulatory standards and WHO’s Prevent, Detect, and Respond strategy as a novel effective tool for detecting falsified vaccines.

Non-toxic approaches to enhance radiotherapy outcomes are beneficial, particularly in ageing populations. Based on preclinical findings showing that high-fibre diets sensitised bladder tumours to irradiation by modifying the gut microbiota, along with clinical evidence of prebiotics enhancing anti-cancer immunity, we hypothesised that dietary fibre and its gut microbiota modification can radiosensitise tumours via secretion of metabolites and/or immunomodulation. We investigated the efficacy of high-fibre diets combined with irradiation in immunoproficient C57BL/6 mice bearing bladder cancer flank allografts. Psyllium plus inulin significantly decreased tumour size and delayed tumour growth following irradiation compared to 0.2

Patients with alcoholism and type 2 diabetes manifest altered metabolism, including elevated aldehyde levels and unusually low asparagine levels. We show that asparagine synthetase B (ASNS), the only human asparagine-forming enzyme, is inhibited by disease-relevant reactive aldehydes, including formaldehyde and acetaldehyde. Cellular studies show non-cytotoxic amounts of reactive aldehydes induce a decrease in asparagine levels. Biochemical analyses reveal inhibition results from reaction of the aldehydes with the catalytically important N-terminal cysteine of ASNS. The combined cellular and biochemical results suggest a possible mechanism underlying the low asparagine levels in alcoholism and diabetes. The results will stimulate research on the biological consequences of the reactions of aldehydes with nucleophilic residues.

Following in the footsteps of genomics and proteomics, metabolomics has revolutionised the way we investigate and understand biological systems. Rapid development in the last 25 years has been driven largely by technical innovations in mass spectrometry and nuclear magnetic resonance spectroscopy. However, despite the modest size of metabolomes relative to proteomes and genomes, methodological capabilities for robust, comprehensive metabolite analysis remain a major challenge. Therefore, development of new methods and techniques remains vital for progress in the field. Here, we review developments in LC-MS, GC–MS and NMR methods in the last few years that have enhanced quantitative and comprehensive metabolome coverage, highlighting the techniques involved, their technical capabilities, relative performance, and potential impact.

Substandard (including degraded) and falsified (SF) vaccines are a relatively neglected issue with serious global implications for public health. This has been highlighted during the rapid and widespread rollout of COVID-19 vaccines. There has been increasing interest in devices to screen for SF non-vaccine medicines including tablets and capsules to empower inspectors and standardise surveillance. However, there has been very limited published research focussed on repurposing or developing new devices for screening for SF vaccines. To our knowledge, rapid diagnostic tests (RDTs) have not been used for this purpose but have important potential for detecting falsified vaccines. We performed a proof-in-principle study to investigate their diagnostic accuracy using a diverse range of RDT-vaccine/falsified vaccine surrogate pairs. In an initial assessment, we demonstrated the utility of four RDTs in detecting seven vaccines. Subsequently, the four RDTs were evaluated by three blinded assessors with seven vaccines and four falsified vaccines surrogates. The results provide preliminary data that RDTs could be used by multiple international organisations, national medicines regulators and vaccine manufacturers/distributors to screen for falsified vaccines in supply chains, aligned with the WHO global ‘Prevent, Detect and Respond’ strategy.

The global population is increasingly reliant on vaccines to maintain population health with billions of doses used annually in immunisation programmes. Substandard and falsified vaccines are becoming more prevalent, caused by both the degradation of authentic vaccines but also deliberately falsified vaccine products. These threaten public health, and the increase in vaccine falsification is now a major concern. There is currently no coordinated global infrastructure or screening methods to monitor vaccine supply chains. In this study, we developed and validated a matrix-assisted laser desorption/ionisation-mass spectrometry (MALDI-MS) workflow that used open-source machine learning and statistical analysis to distinguish authentic and falsified vaccines. We validated the method on two different MALDI-MS instruments used worldwide for clinical applications. Our results show that multivariate data modelling and diagnostic mass spectra can be used to distinguish authentic and falsified vaccines providing proof-of-concept that MALDI-MS can be used as a screening tool to monitor vaccine supply chains.

ADVERTISEMENT RETURN TO ISSUEPREVReviewNEXTIon-Exchange Chromatography Coupled to Mass Spectrometry in Life Science, Environmental, and Medical ResearchJudith B. NgereJudith B. NgereDepartment of Chemistry, University of Oxford, Mansfield Road, Oxford OX1 3TA, U.K.More by Judith B. NgereView Biographyhttps://orcid.org/0000-0003-2288-7215, Kourosh H. EbrahimiKourosh H. EbrahimiInstitute of Pharmaceutical Science, King's College London, London SE1 9NH, U.K.More by Kourosh H. EbrahimiView Biography, Rachel WilliamsRachel WilliamsDepartment of Chemistry, University of Oxford, Mansfield Road, Oxford OX1 3TA, U.K.More by Rachel WilliamsView Biography, Elisabete PiresElisabete PiresDepartment of Chemistry, University of Oxford, Mansfield Road, Oxford OX1 3TA, U.K.More by Elisabete PiresView Biography, John Walsby-TickleJohn Walsby-TickleDepartment of Chemistry, University of Oxford, Mansfield Road, Oxford OX1 3TA, U.K.More by John Walsby-TickleView Biographyhttps://orcid.org/0000-0002-1287-9580, and James S. O. McCullagh*James S. O. McCullaghDepartment of Chemistry, University of Oxford, Mansfield Road, Oxford OX1 3TA, U.K.*[email protected]More by James S. O. McCullaghView Biographyhttps://orcid.org/0000-0003-4733-1205Cite this: Anal. Chem. 2023, 95, 1, 152–166Publication Date (Web):January 10, 2023Publication History Received29 September 2022Published online10 January 2023Published inissue 10 January 2023https://doi.org/10.1021/acs.analchem.2c04298Copyright © 2023 The Authors. Published by American Chemical SocietyRIGHTS & PERMISSIONSACS AuthorChoiceCC: Creative CommonsBY: Credit must be given to the creatorArticle Views760Altmetric-Citations-LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (4 MB) Get e-AlertsSUBJECTS:Chromatography,Environmental science,Food,Ions,Organic acids Get e-Alerts

During peptidoglycan recycling (PR) bacteria can recover extracellular fragments of peptidoglycan (PGN) liberated by peptidoglycan turnover (PT) during cell growth and division, and reuse them in cell wall biosynthesis or central carbon metabolism. In Gram-negative bacteria, PR has been well studied, and functions in the induction of resistance to certain classes of antibiotics, and in host-pathogen interaction. However, while Gram-negative cell envelope architecture allows for highly efficient PR, Gram-positive bacteria, which lack an outer cell membrane and are instead enclosed by a glycopolymer layer, can shed large quantities of PGN-derived material to the external environment during growth. Nonetheless, the occurrence of PR was recently demonstrated in several Gram-positive bacteria, including the Gram-positive bacterial pathogen Staphylococcus aureus , and its potential adaptive functions are largely unexplored. Given the known roles of PR in Gram-negative bacteria, and that Gram-positive bacteria include several important human pathogens, we asked what role PR may play during Gram-positive pathogen-host interaction. Usingthe model insect host Drosophila melanogaster, we demonstrate that S. aureus mutants impaired in extracellular PGN hydrolysis (Δ atl ) and PGN fragment uptake (Δ murP ) show differential virulence compared to their wild-type counterpart. This was linked to increased activation of the D. melanogaster Toll-cascade by spent supernatant from the Δ atl mutant. Thus, we propose that S. aureus , and potentially other Gram-positive bacteria, may use extracellular PGN degradation during PT to simultaneously process PGN fragments for recycling and for immune evasion, while recovery and/or metabolism of peptidoglycan fragments during PR may play more subtle roles in determining virulence. Author summary PGN is a key component of the bacterial cell wall, forming a stress-bearing sacculus surrounding the cell and providing cell shape. During growth and division, the sacculus is dynamically degraded and remodelled to ensure daughter cell separation, resulting in PT. PGN fragments released during PT can be recovered and reutilised by the cell during PR. In Gram-negative pathogens, PR is linked to antibiotic resistance, virulence and modulation of host immune recognition. In Gram-positive bacteria, PR was only recently observed. Here, we explore the roles of PT and PR in host-pathogen interaction in S. aureus, a Gram-positive pathogen of significant clinical relevance. Disruption of PT in S. aureus affected host-pathogen interaction through altering host recognition of shed PGN fragments and PR through modulation of PGN fragment recovery. This improves our understanding of the biology of this important pathogen and may aid development of novel therapeutic approaches to treat S. aureus infections.