Ascaridia galli and Heterakis gallinarum are associated with impaired host performance, particularly in high-performing genotypes. Heterakis gallinarum is also a vector of Histomonas meleagridis that is often co-involved with ascarid infections. Here, we provide a first insight into the alteration of the chicken plasma and liver metabolome as a result of gastrointestinal nematode infections with concomitant histomonosis. 1H nuclear magnetic resonance (1H-NMR) based-metabolomics coupled with a bioinformatics analysis was applied to explore the variation in the metabolite profiles of the liver (N = 105) and plasma samples from chickens (N = 108) experimentally infected with A. galli and H. gallinarum (+H. meleagridis). This was compared with uninfected chickens at different weeks post-infection  representing different developmental stages of the worms.Gut infections of chickens caused by N-oxide, myo-inositol, and acetate. Differential pathway analysis suggested that infection induced changes in (1) phenylalanine, tyrosine, and tryptophan metabolism, (2) alanine, aspartate and glutamate metabolism; and 3) arginine and proline metabolism .A total of 31 and 54 metabolites were quantified in plasma and aqueous liver extracts, respectively. Statistical analysis showed no significant differences (P > 0.05) in any of the 54 identified liver metabolites between infected and uninfected hens. In contrast, 20 plasma metabolites including, amino acids, sugars, and organic acids showed significantly elevated concentrations in the infected hens (P < 0.05). Alterations of plasma metabolites occurred particularly in wpi 2, 6 and 10, covering the pre-patent period of worm infections. Plasma metabolites with the highest variation at these time points included glutamate, succinate, trimethylamine-1H-NMR based-metabolomics revealed significant alterations in the plasma metabolome of high performing chickens infected with gut pathogens—A. galli and H. gallinarum. The alterations suggested upregulation of key metabolic pathways mainly during the patency of infections. This approach extends our understanding of host interactions with gastrointestinal nematodes at the metabolic level.In conclusion, The online version contains supplementary material available at 10.1186/s13099-023-00584-7. Ascaridia galli and Heterakis gallinarum in chickens is of importance, particularly since these species are re-emerging across Europe due to non-cage housing systems that ease completion of parasite life cycle . The data were analysed with one-way ANOVA using the R software version 4.1.2 .0.05 < p ≤ 0.10.For the metabolomics data, statistical significance was determined using student t-test with FDR adjustment. Data from all wpi were either pooled or analysed separately within each wpi to examine the effects of infections on metabolite concentrations. One sample with outlier metabolite results was removed from the plasma metabolite data. Differences were considered significant when p < 0.05 and a tendency for significant difference was declared when http://www.metaboanalyst.ca) was employed for univariate, hierarchical clustering and pathway enrichment analysis of the metabolomics data, and analyses were conducted separately for plasma and liver data, respectively. A hierarchical clustering heatmap showing group averages with Euclidean distance measures and ward linkage was constructed to explore the patterns among the metabolites between the two groups within each wpi. Pareto-scaling normalisation  was applied to metabolite data before analysis. For pathway analysis, data were cross-listed with the pathways in the Gallus gallus Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway library. A global test was selected for the enrichment analysis method, while topology analysis was based on relative-betweenness centrality and scatter plot (testing significant features) was chosen for visualisation method.MetaboAnalyst software  of plasma metabolites at showing significantly higher metabolites in infected hens in wpi 2, 6 and 10.Additional file 4: Figure S2. NMR spectral signal of Trimethylamine-N-oxide from database shown in one-dimensional experiment (1D) data (A) and in two-dimensional (2D) experiment data (B); and from plasma sample of nematode infected hens during wpi 18 shown in 1D experiment (C) and in 2D experiment (D); X axis represents the 1H chemical shift in ppm. The blue colour represents the fitting obtained from Chenomx database, the black line represents the real sample, and the Match Factor is given in percentage (C). Frequency 1 (F1 ppm) represents the 13C chemical shift in ppm, while the frequency 2 (F2 ppm) represents the 1H chemical shift in ppm . Figure S3. NMR spectral signal of Dimethyl sulfone from database shown in one-dimensional experiment (1D) data (A) and in two-dimensional (2D) experiment data (B); and from plasma sample of nematode infected hens during wpi 18 shown in 1D experiment (C) and in 2D experiment (D); X axis represents the 1H chemical shift in ppm. The blue colour represents the fitting obtained from Chenomx database, the black line represents the real sample, and the Match Factor is given in percentage (C). Frequency 1 (F1 ppm) represents the 13C chemical shift in ppm, while the frequency 2 (F2 ppm) represents the 1H chemical shift in ppm . Figure S4. NMR spectral signal of Myo-inositol from database shown in one-dimensional experiment data (1D) (A) and in two-dimensional (2D) experiment data (B); and from plasma sample of nematode infected hens during wpi 18 shown in 1D experiment (C) and in 2D experiment (D); X axis represents the 1H chemical shift in ppm. The blue colour represents the fitting obtained from Chenomx database, the black line represents the real sample, and the Match Factor is given in percentage (C). Frequency 1 (F1 ppm) represents the 13C chemical shift in ppm, while the frequency 2 (F2 ppm) represents the 1H chemical shift in ppm . Figure S5. NMR spectral signal of Serine from database shown in one-dimensional experiment data (1D) (A) and in two-dimensional (2D) experiment data (B); and from plasma sample of nematode infected hens during wpi 18 shown in 1D experiment (C) and in 2D experiment (D); X axis represents the 1H chemical shift in ppm. The blue colour represents the fitting obtained from Chenomx database, the black line represents the real sample, and the Match Factor is given in percentage (c). Frequency 1 (F1 ppm) represents the 13C chemical shift in ppm, while the frequency 2 (F2 ppm) represents the 1H chemical shift in ppm . Figure S6. NMR spectral signal of 3-Methylhistidine from database shown in one-dimensional experiment (1D) data (A); and from plasma sample of nematode infected hens during wpi 18 shown in 1D experiment (B); X axis represents the 1H chemical shift in ppm. The blue colour represents the fitting obtained from Chenomx database, the black line represents the real sample, and the Match Factor is given in percentage (B). Figure S7. NMR spectral signal of 1,7-Dimethylxanthine from database shown in one-dimensional experiment (1D) data (A); and from liver sample of nematode infected hens during wpi 18 shown in 1D experiment (B); X axis represents the 1H chemical shift in ppm. The blue colour represents the fitting obtained from Chenomx database, the black line represents the real sample, and the Match Factor is given in percentage (B). Figure S8. NMR spectral signal of 3-Methylxanthine from database shown in one-dimensional experiment (1D) data (A) and in two-dimensional (2D) experiment data (B); and from liver sample of nematode infected hens during wpi 18 shown in 1D experiment (C) and in 2D experiment (D); X axis represents the 1H chemical shift in ppm. The blue colour represents the fitting obtained from Chenomx database, the black line represents the real sample, and the Match Factor is given in percentage (C). Frequency 1 (F1 ppm) represents the 13C chemical shift in ppm, while the frequency 2 (F2 ppm) represents the 1H chemical shift in ppm . Figure S9. NMR spectral signal of Oxypurinol from database shown in one-dimensional experiment (1D) data (A) and in two-dimensional (2D) experiment data (B); and from liver sample of nematode infected hens during wpi 18 shown in 1D experiment (C) and in 2D experiment (D); X axis represents the 1H chemical shift in ppm. The blue colour represents the fitting of Chenomx database, the black line represents the real sample, and the Match Factor is given in percentage (C). Frequency 1 (F1 ppm) represents the 13C chemical shift in ppm, while the frequency 2 (F2 ppm) represents the 1H chemical shift in ppm . Figure S10. NMR spectral signal of TMAO from database shown in one-dimensional experiment (1D) data (A) and in two-dimensional (2D) experiment data (B); and from liver sample of nematode infected hens during wpi 18 shown in 1D experiment (C) and in 2D experiment (D); X axis represents the 1H chemical shift in ppm. The blue colour represents the fitting of Chenomx database, the black line represents the real sample, and the Match Factor is given in percentage (C). Frequency 1 (F1 ppm) represents the 13C chemical shift in ppm, while the frequency 2 (F2 ppm) represents the 1H chemical shift in ppm .Additional file 5: Figure S11. Hierarchical clustering analysis of plasma metabolites. The heat map of the top 25 most significant plasma metabolites between control and infected groups in at all wpis. The patterns of each compound (shown in each row) were categorized by Ward’s clustering algorithm and Euclidean distance metrics. Increased and decreased metabolite concentration are given in red and blue, respectively.


These species readily react with volatileorganic compounds (VOCs), such as limonene, one of the most abundantcompounds found in indoor enviroments. In this study, reactions ofHOCl/Cl2 with limonene in the gas phase and on indoor relevantsurfaces were investigated. Using an environmental Teflon chamber,we show that silica (SiO2), a proxy for window glass, andrutile (TiO2), a component of paint and self-cleaning surfaces,act as a reservoir for adsorption of gas-phase products formed betweenHOCl/Cl2 and limonene. Furthermore, high-resolution massspectrometry (HRMS) shows that the gas-phase reaction products ofHOCl/Cl2 and limonene readily adsorb on both SiO2 and TiO2. Surface-mediated reactions can also occur,leading to the formation of new chlorine- and oxygen-containing products.Transmission Fourier-transform infrared (FTIR) spectroscopy of adsorptionand desorption of bleach and terpene oxidation products indicatesthat these chlorine- and oxygen-containing products strongly adsorbon both SiO2 and TiO2 surfaces for days, providingpotential sources of human exposure and sinks for additional heterogeneousreactions.The use of household bleach cleaning products resultsin emissionsof highly oxidative gaseous species, such as hypochlorous acid (HOCl)and chlorine (Cl The use of oxidative cleaning agentsand the presence oflimonene lead to subsequent chemical reactions and the formation ofgas-phase products indoors. This study investigates the interactionsof these products and their chemical transformations on indoor relevantsurfaces. The indoor surface-to-volume ratio is approximately by300 times larger than that of the outdoors.26 Therefore, surfaces play a unique role in indoor environments byserving as a reservoir or a sink for adsorption of gas-phase species,31 particle deposition,33 organic film growth,36 and heterogeneous reactions.38 Moreover, compounds that interact and/or react on surfaces can bereleased back into the indoor air.39 Thiscan impact indoor air quality on longer time scales. Hence, studiesof chemical transformations on indoor relevant surfaces are essentialinputs for indoor air quality modeling.26In addition tooutdoor air exchange and gas-phase chemical reactions,a large amount of indoor gas-phase species is removed through surfacepartitioning.2) as a proxy for window glass and rutile (TiO2)as a componentof paint and self-cleaning surfaces, to study surface transformationsduring exposure to indoor prevalent gaseous species generated fromcommon household cleaning products, chlorine bleach, and limonene-containingproducts. Different oxidized and chlorinated VOCs are characterizedby these reactions, and we propose possible reaction mechanisms fortheir formation.In this work, we utilized high surface area silica  placed at the chamber outlet). Thechamber inlet was connected to a glass mixing chamber, which was cleanedand sonicated with methanol  and waterprior to use. All other glassware and vials used in these experimentswere calcined at 500 °C to remove trace organics, and all aqueoussolutions were prepared using Milli-Q water (18.2 MΩ cm). Allexperimental conditions were optimized to overcome the limit of detectionof analytical methods used (UV–vis spectroscopy and gas chromatography–massspectrometry (GC-MS)) so that the signals were distinguished fromthe background noise within the linear range of the assay. GaseousHOCl and Cl2 were generated by bubbling 50 SCCM of zeroair through a 0.36 M aqueous solution of NaOCl  with a pH value adjusted to ∼6.5 usingNaH2PO4·H2O in order to maximize HOCl production .22 It should benoted that the formation of Cl2 gas is unavoidable dueto the slightly acidic precursor pH and Cl–/Cl2 equilibria.25 The gaseous HOCland Cl2 produced were passed through a HEPA filter  to remove aerosols before entering the glassmixing chamber. Limonene vapor was produced by flowing 0.5 SLPM ofzero air into a two-necked round-bottom flask containing 250 μLof liquid (+)-limonene . Additionally, 3 SLPMof zero air was introduced to the mixing chamber as a carrier gas.Thorough mixing of all gaseous species was ensured by using a magneticstir bar at a constant speed before introducing into the blank Teflonchamber for approximately 1 h. All tubing material used in these experimentswas Teflon except the minimal amount of Tygon tubing used for theglassware ports. Prior to all experiments, SiO2  and TiO2 particles  were heated at 200 °C in an oven to remove adsorbed waterand trace organic contaminants as much as possible. Thin films ofindoor relevant surfaces were prepared by sonicating a 75.0 mg/mLslurry of either SiO2 or TiO2 in ethanol  for 20 min, and then transferring 2.00 mL of the slurryto a PTFE dish  uniformly. Allsamples were allowed to air-dry and were placed inside the Teflonchamber at its center position. The prepared thin films were thenexposed to a mixture of gaseous HOCl/Cl2 and limonene for2 h. Gas-phase reaction products were collected after a 2-h exposureusing an impinger containing 10.0 mL of 1:1 methanol/water solution.The impinger inlet was connected to the outlet of the Teflon chamber,whereas the other side was connected to a vacuum. Thin films werethen removed from the chamber, extracted with 1.50 mL of 1:1 methanol/water,sonicated for 1 h, and centrifuged to collect supernatants for HRMSanalysis. All samples were stored at −20 °C and analyzedwithin 24 h of collection.A 240-L environmentalTeflon chamber made of fluorinate ethylene propylene (FEP) film  was used to simulate indoor environments. The schematicof the chamber was previously reported elsewhere.2 and limoneneconcentrations were measured separately by performing two blank experimentsto avoid cross-contamination in analysis. HOCl and Cl2 concentrationsfrom the source were quantified by UV–vis spectroscopy witha pulsed Xe lamp and a diode array detector (Ocean Optics USB 3000)using the maximum wavelengths at 240 and 330 nm, respectively.25 The concentrations of HOCl and Cl2 inside the Teflonchamber were calculated based on total flow rates and the known chambervolume (240 L). For limonene quantification, an impinger was usedsimilar to the previous description. The limonene concentration wasdetermined by GC-MS (Thermo Trace 1300/TSQ 8000 Evo Triple Quadrupole)using external standards .HOCl/Cl2 and limonene exposurewere analyzed using high-resolution mass spectrometry . All samples wereanalyzed in positive-ion mode. The heated electrospray ionization(HESI) source was operated at 100 °C. The ESI capillary was setto a voltage of 3.50 kV at 350 °C. Mass spectra were acquiredwith a mass range of 50–500 Da. Chemical formulas were assignedwith a mass tolerance of <3 ppm with the following element ranges: 12C, 0–10; 1H, 0–30; 16O, 0–10; 23Na, 0–1; 35Cl, 0–2; 37Cl, 0–2. Other peaks with a higher mass tolerancewere not considered in this work.The extracted gas-phaseand surface products after HOCl/Cl31 Briefly, approximately 12 mg of SiO2 (or 20 mg TiO2) particles were pressed onto half ofa tungsten grid  and installed into the IR cell such that the infraredbeam can interchangeably pass through both the pressed sample andthe bare grid in order to collect the surface and gas-phase spectra,respectively. After overnight evacuation, the sample was first exposedto limonene for 30 min at an equilibrium pressure of 77 ± 2 mTorr.Then, all gas-phase limonene in the mixing chamber was evacuated.Limonene and HOCl/Cl2 were not injected at the same timeto avoid back pressure and contamination. Gas-phase HOCl/Cl2 was obtained in a 1.5-L gas bulb by flowing 100 SCCM of zero airthrough a bubbler containing NaOCl precursor. HOCl/Cl2 wasthen introduced into the IR cell to allow for reactions with the surface-adsorbedlimonene and the remaining gas-phase limonene to achieve an equilibriumpressure of 87.1 Torr. Note that zero air was also included in thebulb and the new equilibrium pressure was reached within seconds.The IR cell was isolated after one min of HOCl/Cl2 injection.The equilibrium partial pressures of HOCl and Cl2 insidethe IR cell were approximately 21 and 48 mTorr, respectively. Thereaction was allowed to proceed for another 2 h for equilibration,and then the entire system was evacuated for 1 h. Single-beam spectraof the surface and the gas phase were collected with 250 scans ata resolution of 4 cm–1 over the spectral range extendingfrom 650 to 4000 cm–1. The resulting FTIR spectraof the surface upon adsorption and after evacuation were obtainedby reprocessing with their corresponding background single-beam spectraand subtracting the corresponding reprocessed gas-phase spectra.A complementary experimentto the environmental Teflon chamber experiment was performed usingtransmission Fourier transform infrared  spectroscopy. A custom-made moveable Teflon-coatedinfrared cell (177 ± 2 mL) was connected to a glass mixing chamber(1329 ± 2 mL) with multiple valves for gas injection, two absolutepressure transducers , anda two-stage vacuum system, including a turbomolecular pump (AgilentTwisTorr 74 FS) and a mechanical pump . Moredetails of this FTIR setup have been previously reported.G°) values of all proposed reactionpathways were calculated using Spartan’20 Software (WavefunctionInc.) at the B3LYP/6-311+G** level of theory to confirm the thermodynamicsof the reactions (in the gas phase). The molecular mechanics energyof all molecules was minimized based on the Merck Molecular ForceField.The Gibbs freeenergy  was furtherused to identify fragment ions of a molecular ion and to further investigatethe structure of each product formed. The mass spectrum of gas-phasereaction products between HOCl/Cl2 and limonene , 151.11 (C10H15O+), 133.10 (C10H13+), 105.07 (C8H9+), and 91.05(C7H7+). These mass-to-charge ratiosare similar to the mass spectral data reported in the study of bleachand limonene dark reactions in the gas phase by Wang et al.12 Additionally, a limonene oxidation product wasdetected at m/z 153.13 (C10H17O+) with the following fragment ions: m/z 151.11 (C10H15O+), 135.12 (C10H15+),and 107.08 (C8H11+). The chemicalstructure of this limonene oxidation product at m/z 153.13 was confirmed by comparing with mass spectraof standard compounds (1000 ppm in methanol), including carvone (C10H14O), carveol (C10H16O),alpha-terpineol (C10H18O), and terpinen-4-ol(C10H18O) . It was identified that the mass spectrum of carveol matched thatof the m/z 153.13 compound. Furthermore,we observed compounds with addition of two or more atoms of eitherO or Cl to the limonene backbone, namely, m/z 205.05 (C10H15Cl2+), 211.09 (C10H17OClNa+),and 227.08 (C10H17O2ClNa+). The presence of these compounds indicates that reactions of limonenewith more than one HOCl/Cl2 molecule may occur. A listof compounds detected by HRMS along with their fragment ions is summarizedin 37Cl, were also observed,which verified the assignments of the Cl-containing compounds (Table S1).Following the environmental Teflon chamber experiments, the equilibriummixing ratios of HOCl, Cllimonene 1a reveal2 (2 (m/z 153.13 (C10H17O+), 187.09 (C10H16OCl+), 205.05 (C10H15Cl2+), 221.05 (C10H15OCl2+), and their fragment ions. Therefore,SiO2 and TiO2 surface products are most likelydue to the adsorption of the gas-phase reaction products. Additionally,a minor peak of unreacted limonene (C10H17+) was also observed at m/z 137.13. Proposed formation pathways are discussed (videinfra). All gas-phase and surface products observed in HRMSare summarized in Interestingly, the mass spectra of the surface productsextractedfrom SiO2 1b and Ti2 (2 1c exhibi2 and TiO2 surfaces after exposureto limonene and HOCl/Cl2 followed by a 1-h evacuation collectedat room temperature under dry conditions (RH < 10%) are shown in 2 reaction products on the SiO2 surfacerevealed a few spectral changes compared to the vibrational frequenciesof pure limonene exposed to a hydroxylated SiO2 surface. The FTIR spectra of both SiO2 and TiO2 surfaces showed similar spectral features to those from 2 can react with adsorbed limonene at a surfacelevel without the presence of gas-phase limonene. Most importantly,these surface products remained adsorbed on the surface, even afterovernight evacuation.The mass spectra of the gas-phase reaction productsand the surfaceproducts extracted from the SiO spectra S4 showedvia electrophilicaddition across the double bonds to produce chlorohydrins.26 Dark reactions of limonene with HOCl and Cl2 in the gasphase were previously investigated by Wang et al. and found to producechlorinated limonene.12 Most interestingly,it should be noted again that oxygenated/chlorinated surface productsmay result from adsorption of gas-phase reaction products or fromthe heterogeneous reactions . Here, we proposea reaction mechanism of HOCl with limonene where HOCl undergoes additionacross the double bond of the limonene backbone (Pathway I) to form a chlorohydrin (G1). Due to the availabilityof gas-phase HOCl and the remaining exocyclic C=C bond, G1 may further react with another molecule of HOCl, therebyundergoing another electrophilic addition (III) followedby subsequent dehydrations (IV and V) toform the two C=C bonds in A4.To understandthe chemistry that occurs in indoor environments, several mechanisticpathways see 4 are proPathway II, an oxidized limoneneproduct, confirmedto be carveol (A2) by HRMS, is generated during the reactionsbetween limonene and HOCl. Therefore, a secondary electrophilic addition(VI) can occur, which forms C10H17O2Cl (G2) followed by C10H15OCl (A3) after the loss of water (VII).In 10H14OCl2 (A5). Besides, the FTIR spectra  to form carvone. In addition to HOCl reactions,Cl2 can similarly undergo electrophilic addition to forma dichloroalkane.52 Due to the presence of Cl2 in the gas phase, it can add across an available C=Cbond in a mechanistic pathway similar to that of HOCl (IX), resulting in the formation of A5. Several compoundsformed during gas-phase HOCl and limonene reactions were detectedon the SiO2 and TiO2 surfaces. Similarly, surface-mediatedreactions can occur between limonene in the adsorbed phase with gas-phaseHOCl and Cl2 as discussed earlier. The calculated ΔG° values of all proposed reaction pathways are reportedin Table S2, confirming that these reactionshave negative free energies.An additional surface product with the addition of two Clatomswas observed and was attributed to C spectra 2 showed z 221.05 1, confir53 Oxidative species emitted from cleaningproducts, including but not limited to HOCl and Cl2, alsoreact with various indoor VOCs emitted from these sources. Moreover,the use of two or more cleaning products simultaneously can lead tothe formation of unwanted chemical compounds despite their originalpurpose: cleaning of surfaces. Our experimental results suggest thatthe use of chlorine- and terpene-containing household cleaning productsleads to formation of less volatile compounds. These compounds notonly suspend in the gas phase12 but also adsorb ontoindoor surfaces, effectively increasing their residence times andpotentially participating in additional surface chemical reactions.We reveal that SiO2 and TiO2 are irreversiblyadsorbed by gas-phase products between HOCl/Cl2 and limonene,including C10H16O, C10H15OCl, C10H14Cl2, and C10H14OCl2, which continue to interact with surfacesregardless of evacuation. This result shows that in contrast to ourprevious study of SiO2 with only limonene, where the interactionis reversible,41 these chlorination and oxidationproducts exhibit irreversible adsorption. Most interestingly, heterogeneousreactions can occur between surface-adsorbed limonene and gas-phaseHOCl/Cl2 in which the resulting products adsorb to thesurface with minimal desorption for days. Lower-ventilated spacesusually experience higher indoor volatile compound concentrationsand residence times, thus providing better landscape for heterogeneousreactions to occur. The concentrations of HOCl, Cl2, andlimonene used in our experiments are related to the cleaning activitiesin such low-ventilated indoor settings. Overall, this study showsthat surfaces provide a sink for these chlorinated and oxidized compounds.However, over time, the slow desorption of these surface-bound speciesaffects indoor air quality and provides an additional yet overlookedsource for human exposure. Further studies conducted on longer timescales, under different environmental conditions, and on potentialhealth implications are warranted.In summary, the application ofcleaning products is considereda daily routine for many people around the world. Its significancehas become more apparent ever since the COVID-19 pandemic started.Limonene is one of the most abundant indoor VOCs generated from householdcleaning products and other sources, such as air fresheners and woodproducts.


We detected highly pathogenic avian influenza A(H5N1) clade 2.3.4.4b virus in a domestic cat that lived near a duck farm infected by a closely related virus in France during December 2022. Enhanced surveillance of symptomatic domestic carnivores in contact with infected birds is recommended to prevent further spread to mammals and humans.  On December 27, 2022, the avian influenza National Reference Laboratory of the Agency for Food, Environmental and Occupational Health & Safety in France confirmed a case of highly pathogenic avian influenza (HPAI) A(H5N1) clade 2.3.4.4b virus in a domestic cat. The cat lived with a human family next to a breeding duck farm, which had notified the animal health services of possible HPAI on December 9, 2022, after observing a 20% drop in egg production. After HPAI H5N1 clade 2.3.4.4b virus was confirmed at the farm, 8,375 ducks were culled on December 14. On December 20, the cat displayed disturbances in general condition, including apathy and mild hyperthermia, and was seen by a veterinary surgeon. The cat's condition worsened; pronounced neurologic and respiratory (dyspnea) symptoms appeared, resulting in compassionate euthanasia on December 24. Veterinarians collected sinonasal, tracheal, and anal swab samples after death, and a screening laboratory performed real-time reverse transcription PCR (RT-PCR) targeting the matrix protein and hemagglutinin (H5) genes. The laboratory sent H5-positive tracheal and sinonasal swab samples to the National Reference Laboratory, which confirmed HPAI H5N1 virus by using specific real-time RT-PCR for H5 clade 2.3.4.4b and neuraminidase (N1) genes .https://www.gisaid.org), 20 of which have the E627K mutation, most probably indicating a rapid selection of this mutation in mammalian hosts (We compared the complete sequence of the HPAI H5N1 virus found in the cat (A/cat/France/22P026544/2022) with other HPAI H5N1 virus sequences circulating in France in the same area, including the virus found in the neighboring duck farm (A/duck/France/22P025647/2022). Phylogenetic analyses of HPAI H5N1 genomes indicated that the virus from the cat belonged to the A/duck/Saratov/29-02/2021–like genotype, which has been the predominant virus genotype circulating in France and Europe since September 2022. The cat virus sequence was directly related to virus sequences identified in the same area in December 2022 . FurtherSince winter 2021–22, the number of reported cases of HPAI H5N1 clade 2.3.4.4b infections in mammals has increased  clade 2.3.4.4b virus in domestic cat, France, 2022.


An unconjugated composite peptide vaccine targeting multiple conserved influenza epitopes from hemagglutinin, neuraminidase, and matrix protein and formulated with a safe and highly potent adjuvant, Army Liposome formulation (ALFQ), generated broad and durable immune responses in outbred mice. The antibodies recognized specific epitopes in influenza peptides and several human, avian, and swine influenza viruses. Comparable antibody responses to influenza viruses were observed with intramuscular and intradermal routes of vaccine administration. The peptide vaccine induced cross-reactive antibodies that recognized influenza virus subtypes A/H1N1, A/H3N2, A/H5N1, B/Victoria, and B/Yamagata. In addition, immune sera neutralized seasonal and pandemic influenza strains (Group 1 and Group 2). This composite multi-epitope peptide vaccine, formulated with ALFQ and administered via intramuscular and intradermal routes, provides a high-performance supra-seasonal vaccine that would be cost-effective and easily scalable, thus moving us closer to a viable strategy for a universal influenza vaccine and pandemic preparedness. The current seasonal influenza vaccines are made each year to match the strains expected to be in circulation in the upcoming flu season. This is necessary to circumvent the evolutionary nature of influenza viruses, which undergo routine antigenic drift and shift. The traditional method of influenza vaccine production using embryonated chicken eggs often leads to mismatch between the egg-adapted virus strains and the circulating strains, thereby reducing the protection conferred by the vaccine. Additionally, the process of vaccine production in eggs is time-consuming and does not accommodate pandemic response ,2,3,4. TSeasonal and pandemic influenza both cause severe disease. The spread of highly pathogenic avian influenza virus (A/H5N1) in poultry and its transmission to humans has posed a threat of another pandemic if the outbreaks in poultry are not controlled . AdditioPreviously, we demonstrated that 1 µg of an unconjugated peptide vaccine consisting of highly conserved composite HA, NA, Matrix (M1/M2/M2e) epitopes and a tetanus toxoid T-cell epitope formulated with Army Liposome Formulation with QS-21 (ALFQ) adjuvant elicited robust immune responses in mice when administered via an intramuscular route . Here, wIn this study, we corroborate our previous observation of robust immunogenicity with the low-dose (1 µg) vaccine and further extend our knowledge concerning the durability of immune responses generated via multiple routes of administration and cross-reacting with multiple subtypes of influenza viruses, including the highly pathogenic avian influenza A/H5N1 strains and influenza B strains.Madin-Darby canine kidney (MDCK) cells  were maintained in Eagle’s Minimal Essential Medium (EMEM) , and MDCK-ATL cells (Influenza Reagents Resource (IRR) Manassas, VA, USA) in Dulbecco’s Modified Eagle Medium (DMEM) , with each medium containing 5% fetal bovine serum (FBS) , 2 mM L-glutamine and 1mM sodium pyruvate , 1X non-essential amino acids (NEAA) , 2.5 µg/mL amphotericin B  and 50 µg/mL gentamycin .2. Virus concentrations were determined by the standard tissue culture infectious dose (TCID50) assay on MDCK cells [Several influenza viruses were obtained from the Influenza Reagents Resource (IRR) , established by the Centers for Disease Control and Prevention (CDC), USA. Influenza virus strains used to determine vaccine-induced cross-reactive immunity and neutralization were A/Cal/07/09pdm09 (H1N1), A/Cal/04/09pdm09 (H1N1), A/Mich/45/15pdm09 (H1N1), A/Wisc/505/18pdm09 (H1N1), A/HK/4801/14 (H3N2), A/Vict/361/11 (H3N2), A/Texas/50/12 (H3N2), A/Ind/NIV/06 (H5N1), A/Egypt/321/07 (H5N1), A/Anhui/01/05 (H5N1), A/Vietnam/1203/04 (H5N1), A/Hubei/01/10 (H5N1), A/chicken/Vietnam/16/08 (H5N1), B/NHamp/01/21 (BV), B/OK/10/18 (BY) and B/Bangld/5972/07 (BY)  was used for mice immunization studies. LHNVD-105 has previously been described in detail . It compALFQ was manufactured as described previously ,28,29. BLyophilized Flu Pep11 and Flu Pep5906 were individually reconstituted in Dulbecco’s phosphate-buffered saline pH 7.4 (DPBS) to achieve a concentration of 1.6 mg/mL per peptide. Two different formulations were created: 1 µg dose (low-dose) and 20 µg dose (high-dose).Low-dose or high-dose LHNVD-105/ALFQ vaccines were formulated by adding 4.375 µL or 87.5 mL, respectively, of each reconstituted peptide and 175 µL of ALFQ  to a sterile vial. To equalize the volume, 166.25 µL DPBS was added to the low-dose vaccine. Vaccine formulations were vortexed at a slow speed for 30 s, and then put on a roller for 15 min to complete mixing. The mixed vaccine formulations were then stored at 4 °C for 16 h prior to immunization. Each mouse was injected with 50 µL of low- or high-dose vaccine. The same formulations were used for intramuscular and intradermal administration routes.®) mice, 3–4 weeks of age, were obtained from Envigo  for use in this study, as described previously [Animal studies were carried out in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All animal procedures were approved by the Institutional Animal Care and Use Committee . Female Institute of Cancer Research (ICR-CD-1eviously . The com5 TCID50 per mL) in PBS (100 µL per well) for 18–24 h at room temperature (RT) and 2–8 °C, respectively. Serial dilutions of serum samples starting at 1:100, with five-fold dilutions , were added to antigen-coated plates blocked with 3% normal goat serum  and detected with Horse Radish Peroxidase (HRP)-conjugated isotype-specific goat anti-mouse IgG . TMB Substrate Solution  was added prior to being quenched with TMB STOP solution . The absorbance values (450 nm) of each well were obtained using a SpectraMax Plus Microplate Reader . Data were expressed as endpoint titers defined as the reciprocal of highest dilution that yielded an absorbance value greater than or equal to twice that of the pooled pre-immune serum. Due to a minimum starting serum dilution of 1:100, titers below 100 were reported as nil.Serum anti-influenza levels were evaluated using peptides or live/inactivated influenza viruses, using the procedure previously described . Briefly® Spin-X® centrifuge tube filters , heat-inactivated at 56 °C for 30 min and centrifuged at 8000 rpm (5900× g) for 2 min. Sera were subsequently serially diluted two-fold from the starting dilution of 1:640 in TPCK-trypsin medium in a 96-well plate. Then, 120 µL per well of influenza A viruses (H3N2 or H1N1) was added to each well containing 120 µL of diluted antisera and incubated for 2 h at 37 °C/5% CO2. This addition of an equal volume of virus into the anti-influenza sera samples further increased the antisera dilution two-fold to a starting dilution of 1:1280 and a final dilution of 1:81,920. Confluent MDCK cell monolayers in 96-well plates were washed with PBS  using an ELx405 Automated Plate Washer  10 min before the next step. After 2 h incubation, 100 µL/well of each dilution of antisera-virus mixture was transferred to quadruplicate wells of pre-washed MDCK cell monolayers. Wells containing virus dilutions only and MDCK cells only were included in each plate as positive and negative controls for infectivity, respectively. The assay plates were incubated at 37 °C/5% CO2 for 4 days. After incubation, the plates were stained with Crystal Violet and scored by recording the cytopathic effect (CPE) of the virus on the MDCK cell monolayer. Clear wells representing an absence of MDCK cells were scored as positive CPE and indicated an absence of neutralization of viral replication by the antisera tested. Wells with a blue coloration representing the presence of MDCK cells were scored as negative CPE and indicated neutralization of viral replication by the antisera tested. Neutralizing titers were reported as reciprocal of the highest dilution of serum that showed neutralization of influenza virus in three out of four wells with the MDCK cell monolayer.MDCK cells were seeded for 3–4 days in 96-well plates . Pooled anti-influenza sera samples were diluted 1:640 in plain EMEM medium, added to CostarTM global phylodynamic reconstructions were retrieved from the Global Initiative on Sharing All Influenza Data (GISAID) (accessed on 23 May 2023) [www.ormbunkar.se/aliview) (downloaded on 24 November 2021) and aligned with Multiple Alignment using Fast Fourier Transform (MAFTT) [https://www.megasoftware.net/ and visualized with Tree Figure Drawing Tool . Bootstrap values of 100 were shown. Nucleotide sequences for H5N1 and B/Yamagata strains were unavailable .Publicly available nucleotide sequences of H1N1, H3N2, H5Nx and B/Victoria influenza strains (210 sequences), commercial seasonal vaccines and the EpiFluay 2023) . All seq (MAFTT) . Neighbo (MAFTT)  https://p < 0.05).Data were analyzed using GraphPad Prism 9 . Antisera titers were expressed as mean (±standard deviation) from five mice per group. Two-way ANOVA tests were used to compare immune responses elicited by the low-dose and high-dose vaccines, as well as between intramuscular and intradermal routes of administration, using Šidák correction for multiple comparisons unless otherwise stated  and the high dose (20 µg) per mouse, administered either intramuscularly or intradermally. There was no observed reactogenicity at the injection site . We did 5) a log higher than HA + NA (titers up to 104) between day 42 and day 63 (p = 0.0143) (The high-dose (20 µg) administered either intramuscularly or intradermally generated peak titers to M1/M2/M2e + T-cell epitope (titers up to 10d day 63 . The hig 0.0143) A.p = 0.0107)  (p < 0.0001)  C or intr 0.0001) D, comparIgG2b titers to HA + NA and M1/M2/M2e + T-cell epitope were observed 21 days post primary immunization in three out of the four groups: low- and high-dose intramuscular groups and the high-dose intradermal group . The lowp = 0.0107)  (p = 0.0019) (IgG2b titers to M1/M2/M2e + T-cell epitope showed a profile similar to that obtained with IgG1. The high-dose vaccine demonstrated significantly higher titers than the low-dose vaccine when administered intramuscularly ( 0.0001) C and int 0.0019) D.FigureOverall, the high-dose vaccine demonstrated significantly enhanced antisera responses compared to the low-dose one. Between the two routes of administration, the intramuscular route generated significantly higher titers to the HA + NA composite peptide compared to the intradermal route. However, titers to the M1/M2/M2e + T-cell epitope composite peptide were largely comparable between the two routes of administration.Further expanding the antisera profile analysis beyond day 63, both IgG1 and IgG2b titers were observed to HA + NA and M1/M2/M2e + T-cell epitope up to day 200 post primary immunization . This inp = 0.01)  were comparable between the two doses and the two routes of administration A. Titers = 0.01) B. Notabl = 0.01) A,B demonFurther analysis of isotype-specific IgG responses showed IgG2a and IgG3 titers to both composite peptides HA + NA A,C and MAntisera responses generated by the combination of multiple conserved epitopes of HA, NA and M2e, formulated with ALFQ adjuvant, encompassed four different isotypes of IgG: IgG1, IgG2b, IgG2a and IgG3. As demonstrated in Serum antibody responses to various subtypes of influenza A and B viruses were analyzed by ELISA. IgG1 titers to A/Cal/07/09pdm09 (H1N1), A/HK/4801/14 (H3N2), A/Ind/PR8/06 (H5N1) and B/OK/10/18 (BY) were observed in all four groups at day 49 A,C and dTo demonstrate that the composite peptide vaccine LHNVD-105, formulated with ALFQ adjuvant, generated broadly active and durable antibody responses to influenza viruses, we analyzed pooled sera from day 200 bleeds and observed comparable antisera titers in all four groups of low- and high-dose vaccine administered intramuscularly or intradermally to various strains of human influenza A groups 1 and 2 A, avian Neutralizing antibodies to Group 1 and 2 influenza A viruses were observed in sera from all four groups. The neutralizing titer in pooled sera samples from day 42 and day 63, against a seasonal human A/H3N2 strain, ranged between 5000 and 40,000 A. The nePhylogenetic analyses demonstrated that this vaccine has the potential to induce binding and neutralizing antibodies against human and avian influenza strains, where neighbor-joining trees demonstrated broad antisera cross-reactivity to globally circulating strains of H1N1, H3N2, H5Nx and B/Victoria influenza viruses .Figure Small synthetic peptide vaccines can be easily synthesized and scaled, overcoming the time-consuming process of egg-based production that leads to antigenic changes during manufacture. Multiple conserved epitopes from HA, NA and Matrix (M1/M2/M2e) with a universal T cell epitope are combined to form composite peptide vaccine antigens that promote broad immunity to group 1 and 2 influenza virus strains . In addiAnother aspect to consider for vaccine efficacy is the route of administration. In the standard intramuscular route, a vaccine is delivered into the muscle tissue and subsequently circulates into the draining lymph nodes to be presented to the resident antigen-presenting cells (APC), which in turn activate the B cells and T cells. Other routes of vaccine delivery are being explored due to lessons learned from the COVID-19 pandemic vaccine supply shortages. One example is the intradermal route of immunization, where a vaccine is delivered into the skin and generates strong and long-lasting immune responses from a plethora of skin-resident APCs such as dermal dendritic cells, Langerhans cells and macrophages . The intOur previous publication showed that an ALFQ-adjuvanted composite influenza peptide vaccine comprising multiple highly conserved epitopes of HA, NA and Matrix (M1/M2/M2e) and delivered intramuscularly at a low dose of 1 µg per mouse generated a strong and balanced immune response with cross-neutralizing antibodies against Group 1 and Group 2 influenza viruses . The ALFIn this study, we demonstrate that two doses, 1 µg and 20 µg per mouse, of our multi-epitope composite peptide vaccine formulated with ALFQ adjuvant and administered using two different routes, intramuscular and intradermal, generated strong, balanced, and long-lasting immune responses in mice, with broadly neutralizing antibodies to human A/H3N2 and 2009 swine flu pandemic A/H1N1pdm09 strains. Since variable culture conditions are required for each virus strain, the neutralization assay may need to be optimized for each subtype and emerging influenza virus strains. The antisera recognized several strains of human, swine and avian influenza A and influenza B viruses. The high dose of 20 µg per mouse of the composite influenza peptide vaccine showed significantly higher immune responses to peptide components of the vaccine HA + NA (Flu Pep11) and M1/M2/M2e + T-cell epitope (Flu Pep5906) compared to the 1 µg low dose, given either intramuscularly or intradermally. However, no significant functional differences were observed between the two doses in both binding and neutralizing capacities against various subtypes of influenza A and B viruses up to 200 days post primary immunization, thus indicating that the low-dose (1 µg) vaccine might be sufficient to elicit a robust functional immune response, when administered either through the traditional intramuscular route or the novel intradermal route. Notably, the serum antibodies demonstrated recognition of the zoonotic 2009 swine flu pandemic A/H1N1 strain, the highly pathogenic avian influenza A/H5N1 and the influenza B strains of Victoria and Yamagata lineages. Additionally, strong neutralizing titers were obtained against both seasonal human A/H3N2 and 2009 swine flu pandemic A/H1N1pdm09 influenza strains. Due to non-availability of mouse serum samples, additional microneutralization assays could not be conducted. Phylogenetic analyses demonstrated that this vaccine has the potential to induce binding and neutralizing antibodies against human and avian influenza strains. In addition, others have shown that vaccines that targeted matrix peptides elicited antibodies to H5N1, H1N1 and H7N9 which were protective against lethal challenge with these strains ,39,40. TConsidering the impact of avian influenza outbreaks of the A/H5N1 subtype, with occasional transmission to humans leading to a threat of another zoonotic pandemic, and the prevalence of influenza A/H1N1, A/H3N2 and influenza B strains in seasonal outbreaks among humans and pigs, this composite peptide vaccine formulated with ALFQ adjuvant provides an important strategy for developing a universal influenza vaccine.Our previous publication showed the generation of robust and balanced immune responses to the composite peptide influenza vaccine composed of highly conserved HA, NA and Matrix (M1/M2/M2e) epitopes formulated with ALFQ adjuvant . As an eTherefore, this study further demonstrates broad and durable immune responses to peptides and multiple subtypes of influenza viruses. Induced durable immunity across human, swine, and avian influenza viruses, including A/H5N1, neutralized both seasonal and pandemic strains. Moreover, comparable immune responses to influenza viruses were observed with intramuscular and intradermal routes of administration, thereby providing a prospect for multiple vaccine delivery options, especially during public health emergencies.This unconjugated composite peptide vaccine comprising multiple conserved influenza epitopes formulated with a safe and highly potent adjuvant ALFQ, and administered by intramuscular and intradermal routes, offers an alternative strategy to protein-subunit-based or nucleic-acid-based vaccines by providing a cost-effective, easily scalable supra-seasonal vaccine that generates broad and durable immunity to human and avian influenza viruses, thus moving us closer to a universal influenza vaccine and pandemic preparedness.


Background and Objectives: This study aims to identify the minor allele of the single nucleotide polymorphisms (SNPs) DAB2IP rs7025486, IL6R rs2228145, CDKN2BAS rs10757278, LPA rs3798220, LRP1 rs1466535, and SORT1 rs599839 in order to assess the risk of abdominal aortic aneurysm (AAA) formation and define the linkage among these SNPs. Materials and Methods: A case-control study with AAA patients (AAA group) and non-AAA controls (control group) was carried out in a study population. DNA was isolated from whole blood samples; the SNPs were amplified using PCR and sequenced. Results: In the AAA group of 148 patients, 87.2% of the patients were male, 64.2% had a history of smoking, and 18.2% had relatives with AAA. The mean ± SD of age, BMI, and aneurysmal diameter in the AAA group were 74.8 ± 8.3 years, 27.6 ± 4.6 kg/m2, and 56.2 ± 11.8 mm, respectively. In comparison with 50 non-AAA patients, there was a significantly elevated presence of the SNPs DAB2IP rs7025486[A], CDKN2BAS rs10757278[G], and SORT1 rs599839[G] in the AAA group , while LPA rs3798220[C] was significantly higher in the control group (p = 0.049). A haplotype investigation showed that the SNPs DAB2IP, CDKN2BAS, and IL6R rs2228145[C] were significantly elevated in the AAA group  with minor allele frequencies (MAF) of 25.5%, 10.6%, and 15.4%, respectively. Only DAB2IP and CDKN2BAS showed significantly higher occurrences of a mutation (p = 0.028 and 0.047). Except for LPA, all SNPs were associated with a large aortic diameter in AAA (p < 0.001). Linkage disequilibrium detection showed that LPA to DAB2IP, to IL6R, to CDKN2BAS, and to LRP1 rs1466535[T] had D’ values of 70.9%, 80.4%, 100%, and 100%, respectively. IL6R to LRP1 and to SORT1 had values for the coefficient of determination (2r) of 3.9% and 2.2%, respectively. Conclusions: In the investigated study population, the SNPs CDKN2BAS rs10757278, LPA rs3798220, SORT1 rs599839, DAB2IP rs7025486, and IL6R rs2228145 were associated with the development of abdominal aortic aneurysms. Individuals with risk factors for atherosclerosis and/or a family history of AAA should be evaluated using genetic analysis. DAB2IP (rs7025486), IL6R (rs2228145), CDKN2BAS (rs10757278), LPA (rs3798220), LRP1 (rs1466535), and SORT1 (rs599839) have not yet been investigated in the study population, for which only a few studies have reported on other SNPs and their association with AAA  was detected in AAA patients with arterial hypertension  while a significantly higher occurrence of the SNP LRP1 rs1466535[T] was found in patients with a family history of AAA  when compared to the control group. Obesity was significantly more often associated with AAA development for the genotype mutation of SORT1 rs599839[G] . For LPA rs3798220[C] and IL6R rs2228145[C], there were no significant differences in the occurrence of genotype mutations between the AAA group and the controls.Significant risk factors for a genotype mutation risk of AAA development (minor homozygotes and heterozygotes vs. major homozygotes) were investigated . A higheFor the allele frequency analyses, both alleles of the gene were considered, resulting in 296 alleles for the AAA group and 100 alleles for the control group .DAB2IP rs7025486[A] was present in 17.6% (n = 52/296) of the alleles in the AAA group and in 9% (n = 9/100) of those in the control group. The overall OR between the two groups was 0.464, 95% CI [0.220–0.980], p = 0.040 with an OR < 1, indicating a decreased occurrence of the SNP DAB2IP rs7025486[A] in AAA development (protective exposure). Besides the SNP DAB2IP rs7025486[A], the SNPs CDKN2BAS rs10757278[G], LPA rs3798220[C], and SORT1 rs599839[G] also displayed significant differences in allele frequencies between the AAA and control groups. Here, the OR > 1 indicated the increased occurrence of these SNPs in AAA. The minor allele of CDKN2BAS rs10757278[G] was detected in 25% (n = 74) of male AAA patients and in 3.4% (n = 10) of female patients, with an OR for both sexes of 1.935, 95% CI [1.083–3.454], p = 0.024. The minor allele of LPA rs3798220[C] was displayed in 1.4% (n = 4) of the male AAA patients and was not detected in female AAA patients, but was detected in 3% (n = 3) of the female controls. The OR for both sexes was 3.842, 95% CI [1.011–14.600] in the AAA group with p = 0.049. The SNP SORT1 rs599839 showed a distribution of the minor allele [G] in 10.8% (n = 32) of the male and in 1.7% (n = 5) of the female AAA patients and was significantly higher than in the control group, p = 0.035. The OR for both sexes in the AAA group was 2.714, 95% CI [1.036–7.110]. The allele frequencies of LRP1 rs1466535 [T] and IL6R rs2228145[C] did not show significant differences between the AAA and the control groups for either sex .The minor allele of the SNP DAB2IP rs7025486[A] with a minor allele frequency (MAF) of 25.5% (p = 0.037). In the AAA group, this SNP had a frequency for major homozygote (GG), heterozygote (GA), and minor homozygote (AA) of 70.3% (n = 104), 24.3% (n = 36), and 5.4% (n = 8), respectively, for both sexes. The other two SNPs with significant differences in haplotypes were CDKN2BAS rs10757278[G]  and IL6R rs2228145[C] . The SNPs LRP1 rs1466535[T], LPA rs3798220[C], and SORT1 rs599839[G] showed no significant differences in haplotypes .Regarding zygosity, the similarities or differences between the individuals’ alleles, the set of DNA variations (polymorphisms) adjacent to one another at the same locus that tend to be inherited together (haplotypes), are presented in DAB2IP rs7025486[A] in the AAA group with 29.7% (n = 44) vs. 14% (n = 7) in the control group (p = 0.028) (CDKN2BAS rs10757278[G] showed a mutation in 43.9% (n = 65) of the AAA patients which was significantly higher than in the control group  (p = 0.047). For the SNPs LRP1 rs1466535[T], IL6R rs2228145[C], LPA rs3798220[C], and SORT1 rs599839[G], no significant differences in mutation occurrence were detected .The mutation occurrences in each genetic polymorphism revealed a higher occurrence of the SNP = 0.028) . Also, tDAB2IP rs7025486[A] and CDKN2BAS rs10757278[G] were significantly different in all three investigated parameters: allele frequencies, haplotypes, and mutation occurrences, while no difference was detected for the SNP LRP rs1466535[T]   .p = 0.180 and 0.401) .Aneurysm sac size (>50 mm versus <50 mm) and morphology of AAA (saccular versus fusiform) revealed significant differences between the parameters in all SNPs, except for LPA rs3798220[C] (d 0.401) . With reCDKN2BAS rs10757278[G], IL6R rs2228145[C], and SORT1 rs599839[G] were in accordance with the HWE in the control group  (DAB2IP rs7025486[A] and LPA rs3798220[C] were in accordance with the Hardy–Weinberg Disequilibrium (HWD) in the control group .Regarding the Hardy–Weinberg Equilibrium (HWE), the four SNPs LRP1 rs1466535[T], ctively) . In contLPA rs3798220[C] to DAB2IP rs7025486[A], to IL6R rs2228145[C], to CDKN2BAS rs10757278[G], and to LRP1 rs1466535[T] had D’ values of 0.709, 0.804, 1.00, and 1.00, respectively. Expressed as the correlation coefficient 2r, the two highest 2r values occurred for IL6R rs2228145[C] to LRP1 rs1466535[T] and for IL6R rs2228145[C] to SORT1 rs599839[G] and reached 3.9% and 2.2%, respectively.Linkage disequilibrium (LD) describes the nonrandom association of alleles at two or more loci . The SNPCDKN2BAS rs10757278, LPA rs3798220, SORT1 rs599839, DAB2IP rs7025486, and IL6R rs2228145 are associated with the development of abdominal aortic aneurysms (AAA) in a study population. To our knowledge, this is the first study to investigate polymorphisms in the selected SNP panel and to assess the Linkage Disequilibrium (LD) among them.The present study shows that the single nucleotide polymorphisms (SNPs) of the genes CDKN2BAS rs10757278 +501A>G and IL6R rs2228145 +501A>C) and cholesterol metabolism (LPA rs3798220 +501T>C and SORT1 rs599839 +813A>G) represent risk factors for the development of AAA, while the SNP DAB2IP rs7025486 +501G>A has a protective effect. The SNP LRP1 rs1466535 +504C>T is not associated with AAA in the investigated population.The SNPs in the processes of apoptosis and inflammation , LPA (rs3798220), and SORT1 (rs599839) play a role in cholesterol metabolism  and SORT1 rs599839[G] showed an association with AAA expansion rate [DAB2IP rs7025486[A] and SORT1 rs599839[G] were significantly higher in the AAA group than in the control group. Furthermore, the DAB2IP rs7025486[A] haplotypes and mutation occurrences displayed significant differences. Our findings are in line with another GWAS, which also found significant data for the [A] allele in DAB2IP rs7025486 [DAB2IP is located on 9q33.1-q33.3 and acts as an apoptosis signal-regulating kinase 1-interacting protein. This GTP-ase-activating protein plays a role in mediating TNF-induced cell apoptosis and in cell cycle checkpoint regulation. The latter has an inhibitory effect on vascular smooth muscle cell (VSMC) proliferation via the pathway of JAK-STAT and on endothelial cell migration and angiogenesis [DAB2IP rs7025486[A] and SORT1 rs599839[G] are associated with a large diameter of the aneurysm sac.Recent studies demonstrated that s7025486 . DAB2IP ogenesis . The preCDKN2BAS rs10757278 [G] showed significant differences in allele frequencies, haplotypes, and mutation occurrences and these findings match well with the meta-analysis results of 9p21 (CDKN2BAS) A/G and G/G [CDKN2BAS rs10757278[G] also had a strong D’ with LPA rs3798220[C] (D’ = 100%). This SNP is also significantly associated with metabolic syndrome (MetS) and hypercholesterolemia  and AAA  allele was 2.714 times more likely to occur in the AAA group with a minor allele frequency (MAF) of 2.3%.tabolism ,25. Lu etabolism . A systeLRP1 rs1466535 [T] showed no significant differences in allele frequencies, haplotypes, and mutation occurrences but was associated with a family history of AAA (p = 0.005) and a large aortic diameter. In the present study, the SNP LPA rs3798220[C] showed significant differences in allele frequencies and in the location of AAA but not in the haplotypes nor the mutation occurrence. The present data support the theory that the SNPs LPA rs3798220[C] and SORT1 rs599839[G] but not LRP1 rs1466535 are associated with the development of AAA.In our study, the genetic polymorphism of LPA rs3798220 plays a role in cholesterol metabolism, in which the JAK-STAT signaling pathway is crucial  variant in haplotypes and mutation occurrences, this gene contributed in the linkage disequilibrium to a higher D’ value than the other genetic polymorphisms. The consequence of this is that the presence of LPA rs3798220 could interfere with the presence of the other SNPs, such as CDKN2BAS rs10757278, LRP1 rs1466535, IL6R rs2228145, and DAB2IP rs7025486.Clarke et al. reported that clerosis . Due to l as AAA ,36. AlthIL6R rs2228145[C] had significant differences in haplotypes AC and CC as demonstrated in the downstream effect of IL6R (STAT3) expression , CDKN2BAS rs10757278[G], IL6R rs2228145[C], LPA rs3798220[C], and SORT1 rs599839[G], are associated with the development of AAA disease. The SNP LRP1 rs1466535[T], however, is not associated with AAA disease but is associated with a large aortic diameter in AAA. LPA rs3798220 is linked predominantly to the other investigated SNPs. This study could be used to inform the genetic screening of AAA patients and their families.Five SNPs’ variation in the selected panel, i.e.,


After this article  was publIn response to queries about the mouse models, the corresponding author stated that p204/Ifi204 is the mouse ortholog of human IFI16, and that the gene IFI16 was referred to in error, instead of the gene p204. The corresponding author therefore provided the below updated versions of Figs The corresponding author stated that the antibody used to detect p204 is NBP2-27153 from Novus and has provided an updated The corresponding author provided some clarifying information on the origin of the mouse source: the USP12-deficient (Usp12-/-) mice were generated by Cyagen Biosciences Inc. . The corThe original underlying data to support all results in the article and Supporting Information files are provided here as S1 File(XLSX)Click here for additional data file.S2 File(DOCX)Click here for additional data file.S3 FileTable A. Gene-specific primers used for qRT-PCR. Table B. Antibodies.(DOCX)Click here for additional data file.S4 File(TIF)Click here for additional data file.S1 Fig(A) WT BMDMs were infected HSV-1 for 6 hours followed by immunoprecipitation (IP) using anti-UAF1 or IgG, and immunoblotting (IB) analysis. (B-C) WT and Usp12-/- BMDMs were infected with CMV for the indicated times. p204 expression levels were detected by qPCR (B) or western blot (C). Densitometry quantification of band intensity are presented in the right panel. (D-E) WT and Usp12-/- BMDMs were stimulated with poly(dA:dT) for the indicated times. p204 expression levels were detected by qPCR (D) or western blot (E). Densitometry quantification of band intensity are presented in the right panel. (F)Uaf1fl/fl and Uaf1fl/fl;Lyz2-Cre BMDMs were infected with HSV-1 for the indicated times. p204 expression levels were detected by western blot. Densitometry quantification of band intensity are presented in the right panel. (G-H) WT and Usp12-/-BMDMs were pretreated with STAT1 inhibitor Fludarabine (STAT1 i), and infected with HSV-1 for indicated time. p204 expression levels were detected by qPCR (G) or western blot (H). Densitometry quantification of band intensity are presented in the below panel. (I) p204 IB and K48 and K63 ubiquitination analysis using whole-cell extracts of WT and Usp12-/- BMDMs infected with HSV-1 for the indicated time. Data shown are the mean ±SD. *P < 0.05, and **P < 0.01. Ns, no significant. Data are representative of three independent experiments with similar results.(TIF)Click here for additional data file.S2 Fig(A) WT, Usp12-/-, p204-/- and Usp12-/-p204-/- BMDMs were infected with HSV-1 for 24 hours, and expression of ISG15 and MX1 was determined by qPCR. (B) WT, Usp12-/-, p204-/- and Usp12-/-p204-/- BMDMs were stimulated with IFN-β for 6 hours. Expression of ISG15 and MX1 was determined by qPCR. (C) WT or Usp12-/- BMDMs were transfected with control or expression vector for p204, and infected with HSV-1 for 24 hours. Expression of ISG15 and MX1 was determined by qPCR. Data shown are the mean ±SD. **P < 0.01 and ***P < 0.001 by an unpaired t-test. Ns, no significant. Data are representative of three independent experiments with similar results.(TIF)Click here for additional data file.


Dysregulation of tissue H2S levels is thought to contribute to an ageing phenotype in many tissues across animal models. Whether H2S is altered in HGPS is unknown. We investigated hepatic H2S production capacity and transcript, protein and enzymatic activity of proteins that regulate hepatic H2S production and disposal in a mouse model of HGPS . G609G mice were maintained on either regular chow (RC) or high fat diet (HFD), as HFD has been previously shown to significantly extend lifespan of G609G mice, and compared to wild type (WT) mice maintained on RC. RC fed G609G mice had significantly reduced hepatic H2S production capacity relative to WT mice, with a compensatory elevation in mRNA transcripts associated with several H2S production enzymes, including cystathionine-γ-lyase (CSE). H2S levels and CSE protein were partially rescued in HFD fed G609G mice. As current treatments for patients with HGPS have failed to confer significant improvements to symptoms or longevity, the need for novel therapeutic targets is acute and the regulation of H2S through dietary or pharmacological means may be a promising new avenue for research.Hutchinson-Gilford progeria syndrome (HGPS) is a rare human disease characterised by accelerated biological ageing. Current treatments are limited, and most patients die before 15 years of age. Hydrogen sulfide (H Progeroid syndromes are a set of genetic disorders characterized by a shortened lifespan and early onset phenotypic alterations typically associated with advanced age such as hair loss, frailty, and atherosclerosis . ConsequLMNA gene. A mouse model carrying the equivalent 1827C>T;p.Gly609Gly mutation in the Lmna gene has been developed and is known as LmnaG609G+/G609G+ or simply G609G [2S) levels in various model systems [2S levels in G609G mice under methionine restriction has not yet been measured. G609G mice fed a high-fat diet (HFD) had an average lifespan of 193 days compared to 110 days on regular chow (RC) [The most common mutations in HGPS is the point mutation c.1824C>T;p.Gly608Gly in the human ly G609G . G609G mly G609G –13, 10, ly G609G . In the ly G609G . Diet haly G609G , 17. Methow (RC) . Surprishow (RC) , and imphow (RC) . HFD feehow (RC) . As such2S has recently emerged as a potential effector of various longevity boosting interventions, and elevated tissue H2S levels are associated with long lived mutant mouse models [Therapeutic treatments for patients with progeroid diseases are lacking, with an average life expectancy typically less than 15 years . Currente models –24.2S in HGPS, there is overlap between H2S signalling and the mechanisms that underpin rapamycin, sulforaphane, and vitamin D treatment, as discussed in depth in our recent review [ex vivo H2S production is negatively affected across tissues under HFD feeding, with a reduction in the protein levels of H2S-generating enzyme cystathionine-γ-lyase (CSE) [2S donor compound, mitigated against markers of renal injury caused by HFD-induced obesity in mice [2S and the mammalian target of rapamycin pathway [2S production. However, given the unexpected benefits of HFD to lifespan in G609G, a greater understanding of H2S metabolism within the unique context of G609G mice on HFD may help further our understanding of the role of diet and H2S in progeria.While no studies to date have investigated the role of Ht review . Studiesse (CSE) . Additio in mice . These a pathway , but als pathway . These d2S production capacity was found between regular-chow (RC) fed WT and G609G RC mice (p = 0.041), while a non-significant reduction of approximately 45% was found between WT and G609G HFD mice (p = 0.210). There was no significant difference in H2S production capacity between G609G mice on either diet (p > 0.999). Given the significant reduction in hepatic H2S production capacity in G609G RC mice compared to WT controls, we then investigated whether major components of the enzymatic H2S production and disposal processed were altered  were found to be increased by almost 8-fold in G609G mice irrespective of diet (p = 0.005 for RC and p = 0.007 for HFD). Cbs expression was almost doubled in G609G RC-fed mice relative to WT controls (p = 0.028), but comparable after HFD-feeding between genotypes (p = 0.558). Mpst transcript levels were not different between WT and G609G RC mice (p = 0.216) but were reduced by approximately 30% in G609G HFD mice relative to WT controls (p = 0.050). Cse, Cbs, and Mpst expression were unaffected by diet in G609G mice. We then examined expression levels of enzymes that regulate the oxidation and disposal of H2S. Ethylmalonic encephalopathy 1 protein (Ethe1) expression was significantly reduced in G609G HFD mice compared to WT mice (p = 0.003) but not different in G609G RC mice relative to WT mice (p = 0.525). Thiosulfate sulfurtransferase (Tst) expression was reduced in G609G mice relative to WT mice, irrespective of diet (p = 0.047 for RC and p = 0.027 for HFD). Finally, Suox expression was comparable between G609G and WT mice irrespective of diet (p = 0.128 for RC and p = 0.340 for HFD).Hepatic tissue protein homogenates were analysed by the lead acetate assay . A great2S producing and disposal enzymes were also measured (p = 0.003), p = 0.013) but no significant difference between any other comparison. Neither CBS  nor MPST  differed between groups as shown in p = 0.003), with post hoc testing determining a significant difference between G609G RC and G609G HFD mice (p = 0.013) only. ETHE1 levels (p = 0.746).The protein levels of major Hmeasured . A signi1 levels  did not p = 0.074).Gene expression analysis showed that TST mRNA transcripts were significantly reduced in G609G mice fed either diet compared to WT controls . However2S) appears to be essential for dietary restriction benefits in mice [2S levels decrease with age across model organisms [2S) with age is a conserved phenotype across E. coli, C. elegans, D. melanogaster, and mice [2S production in Hutchinson-Gilford Progeria Syndrome (HGPS) despite the emerging understanding that H2S may be central to longevity and healthy ageing, and the commonalities seen in the signalling modalities of H2S and several established HGPS treatments. In this study, we employed the HGPS mouse model G609G [2S production, the accelerated ageing typical of progeroid mice is associated with reduced hepatic H2S production. Given that G609G mice show lifespan extension on high-fat diet (HFD) compared to regular chow (RC) [2S pathway in these mice under HFD. HFD in WT mice typically shortens lifespan, and has been shown to reduce both plasma H2S levels and hepatic activity of CSE, CBS and MPST [Elevated endogenous hydrogen sulfide  , we alsoand MPST .2S production capacity in G609G mice on RC compared to WT controls and a non-significant reduction in G609G HFD compared to WT controls, indicative of a partial rescue of H2S levels on HFD compared to RC in G609G mice. Cystathionine-γ-lyase (CSE) is the major producer of H2S in hepatic tissues [Cse were significantly elevated in G609G mice on both RC and HFD compared to WT controls. These data may point to a futile compensatory elevation of H2S producing enzymes in response to reduced H2S levels, as previously reported in a rat model of human myocardial infarction [Cse expression can be altered by reactive oxygen species (ROS). ROS, and hydrogen peroxide in particular, are capable of elevating expression of Cse [Cse and Cbs comprise the cytosolic endogenous H2S production pathway in mammals, although there is also a distinct pathway for H2S generation within mitochondria mediated by 3-mercaptopyruvate sulfurtransferase (Mpst). However, Mpst transcripts were not found to be elevated in G609G RC and were in fact reduced in G609G on HFD. In order to provide additional insight, future studies could include additional dietary interventions, for example increasing or decreasing the abundance of dietary cysteine or methionine, to better understand which specific dietary components result in modification of hepatic H2S production in the context of the G609G mutation.We report a significant reduction in H tissues , and trafarction . One potn of Cse . Crucialn of Cse . Cse and2S production we then quantified protein levels of H2S producing and disposal enzymes. CSE protein levels may be controlled at the transcriptional level by repression of Cse gene expression [Cse mRNA transcripts were significantly upregulated in G609G mice on both diets but protein abundance of CSE appeared diet-dependent, being elevated in G609G mice only under HFD feeding. One potential explanation is that the extent of post-translational degradation of CSE differed between groups. Ubiquitination is an established mechanism by which CSE stability is controlled and CSE ubiquitination and degradation is promoted by superoxide anions [2S metabolism and nutrient metabolism [2S-oxidising activity of this enzyme was decreased under HFD. We previously identified TST as a putative antidiabetic enzyme and demonstrated that homozygous TST knock out confers a pro-diabetogenic HFD-like phenotype in C57BL/6J mice [To gain better understanding of the regulation of Hpression . Howevere anions . Indeed,e anions . As suchtabolism , in G609tabolism . This hatabolism . We also/6J mice . These f2S and longevity in model organisms. The data acquired here confirmed some aspects of the relevance of H2S in HGPS but is of course descriptive in nature and raises more questions about the specific mechanisms at play. Studies in primary cell culture from patients and progeria animal models that knock down/out or overexpress CSE would assist in proving a mechanistic role for H2S in the first instance. Lentiviral constructs for the overexpression of progerin in mammalian cells are already established  and cou GYY4137 ) could tpression . Future mutation . These mG609G−/+) were generated on a C57BL/6 background by Prof. Carlos López-Otin  [G609G−/+ female and male mice were obtained from the Otin lab and subsequently bred in the laboratory of Prof. Susana Gonzalo  to generate wild type (WT) control mice and homozygous LMNAG609G+/G609G+ mice, referred to simply as G609G mice hereafter [ad libitum under a 12:12 light-dark cycle. Litter mates of G609G genotype were randomly assigned into one of two diet groups and fed either regular chow (RC) or high fat diet (HFD) immediately post-weaning (approximately 3 weeks of age)- diet composition for diets is provided in n = 4), 100 days  or 150 days . Immediately following death, the liver was collected, snap-frozen in liquid nitrogen and stored at −80°C. Tissue samples were subsequently shipped to University of Glasgow for analysis.Mice carrying the human HGPS single-base mutation  . Heterozereafter . All ani2S levels was performed in liver homogenates according to the previously described method [2S sulfide gas produced during this time collects in the head space between the top of the solution in the well and the lead (II)acetate paper, forming a brown-black substrate on the paper. The amount of H2S generated in each sample was quantified by densitometry analysis of the brown-black substrate (ImageJ).Measurement of Hd method . BrieflyRNA was isolated from liver tissue by addition of 500 μL TRIzol  to sections of liver tissue and homogenized using a glass-glass homogeniser. Samples were moved to screw top Eppendorf and 150 uL chloroform added. Samples were then spun by centrifuge at over 8000 g and the supernatant containing the RNA isolate was taken to a fresh Eppendorf. RNA clean-up was performed using a RNAeasy Mini Kit , including the optional DNase digestion step. First strand synthesis of cDNA was performed by incubating 2 ug of RNA  with 0.333 uL 3 ug/uL Random Primer Mix  in a total volume of 15 uL with RNAse-free water at 70°C for 5 min using a MJ research PTC-200 Peltier Thermal Cycler . Synthesis of cDNA was then performed by adding 10 uL master mix  to the first stand sample and heating to 37°C for 1 hour. Samples were then diluted 1:1 with PCR-grade water and used directly for RT-qPCR. RT-qPCR was performed in a 384-well PCR plate. Each well contained 1 ul cDNA, 0.25 uL 10 mM upper primer, 0.25 uL 10 mM lower primer, 3.5 uL PCR-grade water and 5 uL QuantiFast SYBR green PCR master mix .−ΔΔCt. The endogenous control gene was β2M, which has been previously shown to be an appropriate housekeeping control gene for studies that alter diet [The PCR reaction was performed using a 7900HT Fast Real-Time PCR System  using the following conditions: 95°C for 5 minutes; 94°C for 30 seconds, 60°C for 30 seconds, 72°C for 30 seconds for 40 cycles; 72°C for 5 mins. Fold change was calculated following the ΔΔCt method and expressed as 2ter diet . Primers20) and the membrane was blocked with 5% milk in 1xTBST for 40 min. The membrane was washed 5 times with 1xTBST for 5 mins under constant shaking. Primary antibodies  were added to the membrane in 5% BSA in 1xTBST. Cystathionine-γ-lyase  and ethylmalonic encephalopathy 1 protein  primary antibodies were used at 1:1000 dilution, cystathionine-β-synthase , 3-mercaptopyruvate sulfurtransferase , and thiosulfate sulfurtransferase  were used at 1:500 dilution and total OXPHOS rodent cocktail (ab110413) was used at 1:10000 dilution. Primary antibodies were allowed to incubate with the membrane overnight at 4°C, under constant shaking. Anti-rabbit secondary antibody  was used at 1:10000 dilution in 5% BSA in 1xTBST as the secondary antibody for all blots. The secondary antibody was allowed to incubate with the membrane for 1 hour at room temperature, under constant shaking. The membrane was washed 5 times with 1xTBST for 5 mins, under constant shaking before addition of all antibodies and before imaging by an Odyssey M Fluorescent imager . Protein signals were quantified using densitometry software  and normalised to the total protein signal of their respective lane.Protein lysate was obtained by homogenisation of liver tissue in 1 mL ice cold RIPA buffer  containing protease and phosphatase inhibitors  using a glass-glass homogeniser. Homogenate was left on ice for 40 mins and then spun by centrifuge at over 8000 g for 10 minutes at 4°C. The supernatant was subsequently collected and used as protein lysate. Protein concentration was assessed by BCA assay  and 20 ug of protein was loaded per well into homemade 4–12% bis-tris polyacrylamide gels. Precision Plus Protein™ Dual Xtra Standards protein marker  were added to a well on each gel. Proteins were separated by electrophoresis at 90 V for 90 mins and then transferred onto nitrocellulose membrane at 0.25 V for 1 hour. Membranes were stained with Ponceau-S , briefly washed in deionised water and the resulting total protein stain was captured using a Chemidoc™ XRS System . The Ponceau-S stain was removed by 1xTBST 3/26% HNO3 was added which produced a colorimetric change. Results were quantified by spectrophotometry, measured at 460 nm absorbance. All samples were performed in duplicate, calculating the average absorbance.Thiosulfate sulfurtransferase (TST) rhodanese activity was determined by measuring thiocyanate production capacity as previously described . The asspost-hoc multiple comparison tests. * denotes a p value of < 0.05, ** denotes a p value of < 0.01, and *** denotes a p value < 0.001.All statistical analyses were performed using Prism 9  software. Data were first analysed by Grubbs outlier test with alpha set to 5%. Any outliers identified are noted in figure legends. Statistical significance was determined by performing non-parametric Kruskal-Wallis tests comparing WT, G609G RC and G609G HFD groups with Dunn’s correction for Supplementary Figure 1


Staphylococcus aureus is a major human and animalpathogen, responsible for high levels of morbidity and mortality worldwide.The intracellular survival of S. aureus in macrophages contributes to immune evasion, dissemination, andresilience to antibiotic treatment. Here, we present a confocal fluorescenceimaging assay for monitoring macrophage infection by green fluorescentprotein (GFP)-tagged S. aureus as afront-line tool to identify antibiotic leads. The assay was employedin combination with nanoscaled chemical analyses to facilitate thediscovery of a new, active rifamycin analogue. Our findings indicatea promising new approach for the identification of antimicrobial compoundswith macrophage intracellular activity. The antibiotic identifiedhere may represent a useful addition to our armory in tackling thesilent pandemic of antimicrobial resistance.Antimicrobial resistance has emerged as a global publichealththreat, and development of novel therapeutics for treating infectionscaused by multi-drug resistant bacteria is urgent.  It was thought that this process wouldbe similarly successful in discovering antibiotics. However, severalattempts to use HTS against large corporate combinatorial librarieshave failed to identify new broad-spectrum antibiotics.Staphylococcus aureus is a leadingcause of hospital and community acquired infections, and its abilityto develop resistance to antimicrobial agents makes it a priorityfor the development of agents with new modes of action, superior toxicityprofiles, and intravenous (IV)/oral switch administration. During S. aureus pathogenesis, the interaction with hostmacrophages has a pivotal role in determining the outcome of infection. S. aureus has evolved multiple strategies to survivewithin, manipulate, and escape from macrophages.10 The macrophage intracellular environment not only shieldsthe bacteria from most antimicrobials but also can support the developmentof persister cells with an antimicrobial tolerant phenotype.12 To address these issues, we developed a high-content human macrophageinfection assay , for the ability to inhibit intracellular S. aureus growth and survival inside macrophages.Our assay (S. aureus (MRSA) (USA300 LAC-GFP).13 THP-1 cellsare human monocytic cells that are differentiated into macrophagesusing phorbol myristate acetate (PMA). These cells present an inflammatorymacrophage phenotype, are highly phagocytic,14 and have been extensively used to study bacterial-macrophage interactions15 and antimicrobial intracellular activity.16 Despite some differences when compared with primary cells,17 the use of this cell line allowed us to overcomeissues with availability, donor variability, and ethical considerationsassociated with primary human blood monocyte-derived cultures, aswell as high reagent costs, and the time consuming protocols of preparingmacrophage-like cells from human embryonic stem cells.18 The differentiated, adherent THP-1 cells wereinfected with USA300 LAC-GFP S. aureus at a multiplicity of infection (MOI) of 10 for 1 h, and to testthe intracellular activity of the compounds, the extracellular bacteriawere killed by incubation with gentamicin prior to the addition ofthe test fractions.To address the failure of conventional screening methodsto identify new antibiotics from drug-like small molecule chemicallibraries, we developed a new ur assay 1a utilizS. aureus stably expressing GFP.Natural product fractions were added tothe cells at 25 and 5 μg/mL and incubated for 16 h. Subsequently,cells were fixed with 4% paraformaldehyde (PFA) and stained with Hoechst33342 (nuclei) and Wheat Germ Agglutinin Alexa Fluor 555 Conjugate(plasma membrane) giving a three-color read out with This assayhas four possible outcomes: inactive test fraction,active but cytotoxic, extracellular active, and intracellular active1a–c.As 19 A panel of 108 test fractionswas selected thatcontained ∼10 compounds per fraction at a concentration of10–50 mg/mL in DMSO. Here, the goal was to complete the entirediscovery effort with less than 1 mL of each test fraction and finishwith a characterized active compound. To achieve this, we needed todetermine the limits of this screening effort and began by screening100 μL aliquots of these fractions. From each fraction, a 500μL aliquot was saved for compound purification. Next, we turned to chromatographically fractionatedmarine microbial fractions (test fractions) developed in the Fenicallaboratory from a large repository of marine microbes .Table 1). Each band was submittedto nanoscaled-NMR analyses and recollected after use. Half of theremaining material was resubmitted to the assay, while the other halfwas saved for further purification. Application of the assay to thefractions identified Y014F9-6 (NMR spectrum provided in With a hit fraction (Y014F9) identified, we turned to methods scaledat the nanomolar level  to conduct the assay-guided fractionationdirectly from the screening aliquot. A sample of the DMSO stock offraction Y014F9 (100 μL) was dried by airflow and redissolvedin MeOH (50 μL). A 50 μL solution of the MeOH stock wasseparated using a 5 cm × 20 cm, pTLC plate (250 μm) (eluent1:9 mixture of MeOH:EtOAc) into eight bands (Y014F9-1 to Y014F9-8). Further purification of this materialby prep-TLC  afforded 520 μg (estimated byNMR) of rifamycin analogue 1 . While database searching  failed to produce hits, we were rapidly able to build twofragments by interpretation of 1H-1H gCOSY and 1H-13C HSQC NMR data , four oxygen substituted methines and four methine-attachedmethyl groups. Furthermore, the 1H-1H gCOSYand 1H-13C HMBC data showed additional correlationswith a trans-disubstituted olefin at C28–C29with a 3JHH coupling constantof 12.7 Hz. Taken together, these NMR data suggested that 1 was a member of the rifamycin subclass of the ansamycin family.22The ansamycin family of polyketides1H-1H gCOSY, 1H-13C HSQC, 1H-13C HMBC,and 1H-1H NOESY) for rifamycin B  to those observed in rifamycin B  and rifamycin S , therein indicating that the olefin residue observed by 1H-1H gCOSY  were used to identify the functionalityin 1 that was different for that in rifamycins B or S.Overall the chemical shifts of 1 shared a strong similaritywith selected shifts for rifamycin B and rifamycin S (Supporting Table 5). The aromatic proton H3 was not present, which indicated functionalization (commonlyobserved as oxidation) at C3. Functional modification between C25 and C28 was suggestedby chemical shift differences observed for H25, H27, C25, C27, andC28. Using these data as a guide, we were able to determine that 1 had secondary alcohols at positions C25 and C27, was notacetylated at C25 as observed in rifamycin B , and was not O-methylated at position C27 .In addition to these structural differences were not consistent with the same glycolic acid present at C4 in rifamycinB, as noted by the lack of signals seen for rifamycin B (H38 δ4.74 and C38 δ 67.7 ppm). In contrast, 1 containeda 2-propanone functionality whose position was tentatively assignedto C4 . This was further supported by thepresence of cross peak duplication in the 1H-13C HMBC spectrum, and exchange peaks in the 1H-1H NOESY spectrum suggesting that at least two conformations werepresent.Chemical shift analyses . This observation was further supported by the increasein the hydrolysis of 1 to 2 upon storagein CD3OD solution during NMR studies (data not shown).While methods were established to enable assignment of the aromaticresonances for rifamycin B (Supporting Table 3) and rifamycin S (Supporting Table 4),complete assignment of the aromatic resonances of 1 wasnot possible. The data reported in Supporting Tables 3 and 4 were collected for samples of rifamycin B orS at comparable concentrations as 1 in CD3OD (note the inclusion of 13C NMR spectra and peaks withinthe aryl region of the 1H-13C HMBC for thesetwo compounds).While the polyketide synthase (PKS) derived region(C15–C29and C30–C34) remained uniform, we soon discovered that thealkoxy-2-propanone (C35-C37 in 1 by interpretation of 1H-1H NOESY NMR data (Table 2). Fortunately, a wealth of X-ray crystal structures of rifamycinanalogues exists in the CDCC database. Using the crystal structureof rifampicin methanol solvate trihydrate (deposition CDC 859793),we systematically compared the observed NOEs  and coupling constants  with the configuration observed inthe crystal structure data  and low oxygen recoveryassay (LORA),251 demonstratedactivity against M. tuberculosis withMIC values of 1.3 and 16.8 μM, respectively. Parallel MABA analysesindicated that this compares favorably to rifamycin O (0.093 μMLORA), rifamycin SV (0.044 μM LORA), and rifampicin (0.13 μMLORA or 0.024 μM MABA). While active, the reduced activity of 1 is not unexpected given its need for oxidative activationto 2 .S. aureus developedand demonstrated, the stage isset for larger high-throughput screens of small molecule librariesand for the identification of novel natural products from extractscreening. With the ultimate need to develop new antibacterial drugsto tackle the global AMR crisis, we expect that this protocol willbe adapted for general use as it allows the identification of compoundsthat can target intracellular bacteria. As described herein for S. aureus and by Brodin and Christophe5 for M. tuberculosis, the use of intracellular assays offers an important next step inidentifying cell viable, and hence active, hits.With the first three-color intra-macrophageinfection assay for 26 many of whichhave demonstrated clinical utility.27 Accessto sequence data and new genomic tools has revolutionized our abilityto analyze and manipulate large complex biosynthetic gene clusters(BGCs).28 This capability has advancedto the point where it is now possible to refactor old drugs for bettertreatments of infectious diseases.29 Here,we applied a new screening approach to an unexplored, marine rifamycinproducer, S. arenicola,20 to gain access to a new class of rifamycin analogues.32 On-going sequencing efforts on strains of Amycolatopsis and Salinispora illustrate the complex divergencewithin the biosynthesis of rifamycin observed in strains of Amycolatopsis and Salinispora . While the PKS portion of this pathwayretains a common overall gene architecture , a high degree of divergence is apparentwithin the pre-PKS supply  and post-PKS tailoring enzymes . This divergence suggests that other, yet unexplored,rifamycin analogues, particularly those obtained through post-PKSmodifications, could also exist.20 Successfulcapture of these modifications through comparative genomic analysesprovides an excellent opportunity to blueprint a minimal rifamycinsynthase with the goal of delivering new clinical agents. Here, thetremendous wealth of Salinispora genomes providesan excellent foundation to launch such efforts.33Marine actinobacteriaare known for their prolific production ofsecondary metabolites,34 leprosy,35 and Traveler’s diarrhea.36 Rifamycin polyketides are one of the few drug classes activeagainst replicating and non-replicating bacteria, and new leads arecritical to further clinical improvements. A recent survey of a seriesof strains of S. arenicola  indicated that extracts from 10 selectedstrains produced rifamycins, whose structure and functionality hasyet to be explored. Here, we demonstrate how the combination of anew assay and miniaturized isolation methods can effectively directidentification of new natural products as starting points for thedevelopment of new drugs.The rifamycin group of antibiotics has demonstratedefficacy againstbacterial and mycobacterial infections marked by its critical applicationsin the treatment of tuberculosis,2 environment. Preparation of THP-1 cells priorto infection, THP-1 monocytes were differentiated into macrophagesby treatment with 50 nM phorbol 12-myristate 13-acetate for 3 days at a density of 5 × 104 cells per wellin a 96-well tissue culture plate .The media was then replaced with fresh PMA-free media, and the cellswere left to rest for 24 h before the infection protocol. Cell differentiationfollowing PMA stimulation was monitored over time by microscopic observationof cell morphology and plastic adherence properties.THP-1 cells (kindgift from Prof. David Hume) were maintained in RPMI-1640 medium (ThermoFisher) supplemented with 10% heat inactivated fetal bovine serum and GlutaMax (Thermo Fisher) at 37 °Cin a 5% COS. aureus USA300 LACstrain that constitutively expresses GFP under a sarA-P1 promoterinserted markerless in the bacterial chromosome14 was used in this study. Bacterial overnight culture intryptone soy broth  was sub-cultured in fresh TSB (1:100dilution) at 37 °C, 200 rpm until reaching an OD600 value of 0.6. The bacteria solution was pelleted, washed, and suspendedin macrophage culture media to a concentration of 5 × 106 bacteria/mL. A 100 μL aliquot of the bacterial solutionwas added to the THP-1 macrophage culture, and the plate was centrifugedat 300 × g for 5 min to facilitate the macrophage–bacteriainteraction. The infection was maintained for 1 h at 37 °C ina 5% CO2 atmosphere. Afterward, the media was replacedwith media containing 100 μg/mL of gentamicin and left to incubate for 30 min to kill extracellular bacteria. Thesupernatant was then removed, and the cells were washed twice withfresh media prior to addition of the test fractions.The 1.A total of 108 marine microbialtest fractions containing ∼5 mg/mL of dry fraction in DMSO(Sigma–Aldrich) were screened. For the screen, each fractionwas diluted in macrophage culture media at 200× and 1000× and added tothe thoroughly washed THP-1 macrophages at total volume of 200 μL/well,and the plate was left in the incubator overnight (16 h). Equivalentamount of DMSO was added to the cells in the DMSO control wells. Comparablemethods were applied to test fractions and purified samples of THP-1 macrophages were thenfixed with 4% formaldehyde  in phosphate buffered saline (PBS) for 20 min at 23 °Cand subsequently washed thoroughly in PBS. Next, the cell membranesand the nuclei of the THP-1 macrophages were stained with Alexa Fluor555 conjugated wheat germ agglutinin (ThermoFisher Scientific) andHoechst 33342 (ThermoFisher Scientific) in PBS according to the supplier’sprotocols. The cells were then washed twice thoroughly in PBS, a finalvolume of 100 μL of PBS was added to each well, and the platewas stored at 4 °C until imaging.Imaging was performed using the OperaHCS (PerkinElmer) instrument. Two independent exposures with two fluorescencechannels each were used to avoid bleed through. Exposure 1 parameters:laser excitation 488 nm at 2.5 mW, emission filter 520/35 nm. Exposure2 parameters: laser excitation 561 nm at 2 mW and emission filter585/40 nm and UV excitation at 365 nm and emission filter 450/50 nm,respectively. The exposure time for the three laser channels was 240ms, and the UV exposure time was 20 ms. All imaging was performedusing a 20× Air LUCPLFLN objective (Olympus) with a NA of 0.45,and 20 images per well were captured.S. aureus infected THP-1 macrophages and non-infectedcells . Briefly, nuclei were detected using the algorithm H in the Hoechstchannel, and border objects were removed to ensure only whole nucleiwere analyzed. Area, roundness, and intensity were calculated to filterobjects downstream of the analysis. The cytoplasm was detected usingalgorithm B in the macrophage marker (cytoplasm channel). Those areaswere kept as regions of interest to define individual cell areas.For those regions, properties such as area and intensity and theirstandard deviations were calculated. A “find spots”algorithm was utilized for the infectious bacteria (S. aureus GFP channel) foci analysis. Staining intensityand morphological properties were calculated in all channels, andinfectious ratio  was calculated for thecells identified (Supporting Table 6).Potential artifacts in the analysis were removed using cell-like propertyfilters. The remaining cell-like objects were transferred into a CSVoutput file. Final data analysis and graph plotting were performedin Excel and Prism (GraphPad).Image analysis was completed usingthe image analysis software Acapella (PerkinElmer). Two versions ofthe script were developed, one that is compatible with the Opera instrumentto run while imaging (version 2.7) and one supporting instrumentswith Harmony and Windows 10 (version 5.2+). The script was calibratedusing different treatment conditions: positive (50 nM rifampicin)and negative control (no rifampicin) treated wells, of edcells 1c.The w1) whereas in the remaining wells, there were no live macrophages.Hence, the number of remaining live macrophages followed a stochasticdistribution making the assay not useful. Therefore, we chose MOIof 10 to obtain a stable assay. To further characterize the assay, S. aureus infected and non-infected differentiatedTHP-1 cells were kept overnight in complete RPMI-1640 medium withand without 50 nM rifampicin  and applied to a pTLC plate (5 cm × 20cm) and eluted with a 1:9 mixture of MeOH:EtOAc to deliver three fractionsY014F9-6A, Y014F9-6B, and Y014F9-6C. The second repetition assay identifiedY014F9-6B as the most active fraction.1H NMR spectra werecollected on a sample of each fraction. Comparable methods were usedto screen the activity of purified 1.A 100 μLsample of the test fraction in DMSO was dried by air flow, redissolvedin MeOH (50 μL), applied to a pTLC plate (5 cm × 20 cm),and eluted with a 1:9 mixture of MeOH:EtOAc. Eight bands (Y014F9-1to Y014F9-8) were obtained and submitted to NMR analyses. Each ofthese fractions was rescreened to identify Y014F9-6 as the activefraction. S. arenicola CNY-014strain was cultured in 5 L scale for 7 days with shaking at 180 RPM.After this period, the cultures were extracted with EtOAc (2 ×5 L) and the extract was dried with Na2SO4 andconcentrated by rotary evaporation to yield 400 mg of organic extract.The ethyl acetate extract (400 mg) was subjected to silica chromatography,using a step gradient solvent system of n-hexane,EtOAc, and MeOH  to yield 10 fractions as provided in Supporting Table 1.1 was obtained from the CNY014-S10fraction by pTLC purification. The S10 fraction was dissolved in 100μL of MeOH, applied to a pTLC plate (5 cm × 20 cm), andeluted with a 1:9 mixture of MeOH:EtOAc. Six bands (CNY014-S10A toCNY014-S10F) were obtained. Compound 1 was found in fractionCNY014-S10E. Repetition of the pTLC purification eluting with a 1:10mixture of MeOH:acetone provided pure 1 (580 μg).This material was used for NMR analyses and structure elucidationstudies. Samples of rifamycin B and rifamycin S  were obtained commerciallyto assist in the structure elucidation effort.Pure H 7.26 and δC 77.0for CDCl3 and δH 3.31 and δC 49.0 for CD3OD). The NMR spectra were processedusing Mnova 11.0 (Mestrelab Research) or TopSpin 3.6 (Bruker Biospin)software.NMR data wereacquired with a Bruker Avance III 600 MHz spectrometer equipped witha 1.7 mm cryoprobe. Chemical shifts were referenced using the correspondingsolvent signals (δ