The possible reasons behind thisinteresting insulation performance and the mechanisms leading to theunderlying gel structure are discussed in depth. A focus is made onthe different gelation modes happening between the RMF and silicaphases, from a coating of silica surfaces with RMF species to discontinuousRMF particles within a silica backbone and a continuous RMF backbonewith isolated silica particles. The implications in terms of mechanicalproperties and thermal conductivity are elaborated upon. The initialratio of silica–RMF species in this ethanol-based synthesisaffects the micro- and macrostructure of the composites, resultingin materials with drastically different pore structures and thus aninteresting array of possibilities for a new class of silica-organiccomposite aerogels, based on a sol–gel process.Here, we report thegelation and supercritical drying of ethanol-basedsilica–resorcinol–melamine–formaldehyde (RMF)composite aerogels with relative concentrations of initial reagentsranging from neat silica to neat RMF alcogels. The as-prepared materialsare subsequently supercritically dried with carbon dioxide. Theirproperties include a thermal conductivity in the 15–20 mW·m The motivation behind exploring these differentchemistries for aerogels is about elaborating interesting and innovatingmicrostructures, chemistries, properties, and applications. The varietyof their chemistry allows aerogels to be promising candidates forapplications in thermal insulation,15 catalysisand gas separation,16 oil spillage absorption,17 and so forth.20Aerogels are a classof materials sometimes described as a newstate of matter because their properties differ from those of equivalentbulk materials due to their nanostructured backbone and porosity andtheir high porosity . Aerogelswere first reported by Kistler in 193221 Further downthe road, these porous materials have been explored in detail34 and optionally can be doped with heteroatoms like nitrogen, forexample, through the addition of urea37 or melamine,40 that is, effectivelybecoming resorcinol–melamine–formaldehyde (RMF) resins.Both silica and RF resins can be colloidal with a shared solvent system;for example, both can be synthesized in ethanol–water mixtures,41 which points toward a potential compatibilityof the systems, yet there are only a few examples of their compositeaerogel-related materials in the published literature.48 We hypothesize that an aerogel composite of silica and RMF can providean interesting compromise between acceptable mechanical properties(one of the biggest drawbacks of silica aerogels) and a retentionof silica aerogels’ exceptional thermal insulation properties.Indeed, their unique mesoporous microstructure49 allows them, through the Knudsen effect, to reach an extremelylow thermal conductivity (λ = 12–15 mW·m–1·K–1 STP)—about half of that of standingair (λ ≈ 26 mW·m–1·K–1 STP).Resorcinol–formaldehyderesin (RF) is a particularly interestingcandidate material and was first reported as an aerogel in a seminalpaper by Pekala in 1989.versus observed properties are discussed, and unexpectedresults and their possible causes are explored in detail.In this work, we explore a silica–RMFcomposite aerogelsystem with different proportions of each precursor. Synthesis conditionswere fixed except for the silica–RMF ratio, allowing for adirect comparison of the effect of chemical composition on the microstructureand bulk properties for a wide array of compositions. The hybrid aerogelsare then scouted for performance by measuring their compressive strengthand thermal conductivity under ambient conditions. The expected wt aqueous solution stabilized with 10–15%wt methanol),resorcinol (ReagentPlus 99%), and melamine (99% purity) were all purchasedfrom Sigma-Aldrich and used as-received without further purification.Ammonium hydroxide (ACS reagent 28.0–30.0% NH3 basis)was also purchased from Sigma-Aldrich and diluted to 5.5 mol·L–1 in deionized water before use. Absolute ethanol denaturedwith 5% isopropanol was used as the primary solvent for the synthesis.Tetraethylorthosilicate (TEOS) was purchasedfrom Evonik (Germany), and formaldehyde , is described in detail in our previous publication.41 Briefly, melamine is dissolved in a water–ethanol–formaldehydesolution at 55 °C, and resorcinol is dissolved in ethanol alsoat 55 °C. The two sols are brought together and catalyzed bythe addition of ammonia. For each experiment, the RMF and silica solsare mixed in pre-determined ratios. The ratio between the two precursorsols was selected such that the nominal silica content for 5 h, followed by the release of CO2 at 50 °C.After 15 min of stirring, ammoniumhydroxide was added as a catalystto reach a final ammonia concentration in the mixed sol of 0.128 mmol·LEnvelope densitymeasurements werecarried out on a powder displacement device with a consolidation force of 4.0 N. On each sample, 10 consecutivemeasurements were realized, and the average value was retained. Measurementswere repeated on samples from 5 different monoliths of each composition,for a total of 50 measurements. Given the low intrinsic error on thismeasurement with the type of material under investigation, the reportedvariation is the calculated standard deviation.Skeletal density was measured by heliumpycnometry  using a pressure of 134.447kPa for purging and measurement cycles. Fifty purges followed by 20measurements were realized on each sample, and the average value wasretained. Measurements were repeated on samples from 5 different monolithsfor each composition, for a total of 100 measurements. The reporteddeviation is the calculated standard deviation for these measurements.–2 mbar. Accessible pore volumes and surfaceareas were measured using both classical analytical models  and state-of-the-artnonlocal density functional theory (NLDFT) kernels, as recommendedby the ISO-15901-3 standard.52 For NLDFTcalculations, a cylindrical geometry was assumed for mesopores, basedon Tarazona’s work.54Nitrogen sorption isotherms wererecorded at 77.4 K . Before each measurement,samples were degassed at 105 °C for 20 h at a pressure of 1.3× 102 and 80% N2) with a heating rate of 10 K·min–1 .Thermogravimetric analysis(TGA) curves were recorded under a reconstituted air atmosphere  was conducted on a Bruker spectrometerin attenuated total reflectance (ATR), using a diamond crystal, forwavenumbers ranging between 500 and 4000 cm–2 mbar. The C and H contents weredetermined by infrared spectroscopy. The N content was determinedby measuring the thermal conductivity of the gas phase. The remainingweight was attributed to Si and O contents, assuming the absence ofquantitative impurities. Measurements were carried out with an accuracyof ±0.3%.Elemental analysis was carried out on a LECO TruSpec Micro. Before analysis, sampleswere dried in nitrogen sorption tubes overnight at 105 °C and1.3 × 1055 with monolithic samples. Measurements were carried at least threetimes per composition with a relative error of 12%.Thermal conductivitywas measuredon a homemade guarded hot plate deviceMechanical properties were evaluatedthrough uniaxial compression tests performed on cylindrical samplespreemptively flattened on both ends by using sandpaper, using a universalmaterials testing machine  equippedwith a 2 kN force transducer cell .The compression rate was set to 1 mm/min, and the stress/strain responsewas measured until mechanical failure of the samples. The elasticmoduli were calculated from the slope of the initial linear phase(3–5% strain) of the compression curve.1H, 100.6 MHz for 13C, and 79.5 MHzfor 29Si with 7 mm-diameter zirconia rotors and at a magicangle spinning (MAS) rate of 4 kHz. 1H–29Si and 1H–13C cross polarization MASNMR spectra were acquired to boost the sensitivity, with relativelylong contact times  to minimize the dependencyof the spectral intensities on the 1H–X distance,but the spectra can only be considered in a semi-quantitative approachat best.Solid-state nuclear magnetic resonance(NMR) spectra were acquired on a Bruker AVANCE III system using awide-bore 9.4 T magnet, corresponding to Larmor frequencies of 400.2MHz for Transmission electronmicroscopy (TEM) pictures were acquired on a JEM2200FS JEOL microscopeat an operation voltage of 200 kV.56 Pictures were taken with a smartphone in a roomwith neutral white light.Macroscopic colorimetry ofsamples was assessed on a Pantone uncoated colorimetric scale.41 can both formwet gels in ethanol when catalyzed by ammonia. By finding the optimumNH3 catalyst concentration, composite silica–RMFalcogels of homogeneous appearance were obtained for nominal silicaprecursor contents spanning the entire range from 0 to 100%nom (nom), while gels with a high nominal RMF content (≤48%nom SiO2) did not shrink right after gelation—theybehaved like RMF alcogels in this respect. The color of the exchangedsolvent was highly dependent on the composition, ranging from transparentto deep orange with discernible particles, indicating that reactantswere partially washed out (see the Supporting Information)—leading to potential differences betweenthe nominal concentration of the reactants and the final aerogel composition.The quantification of the solid mass in the exchange solvent by measuringchanges in solvent density was unsuccessful as calculated mass losseswere within the error range of the measurement.Mixing two chemicalsystems requires finding compatible conditions for them to interact.Silica sols and RMF resins 100%nom 1. Gelatinom SiO2 had a visible shine and a smooth, quicklydrying surface, as is also observed for pure silica gels, while sampleswith 10%nom SiO2 or less showed a mat surfacewith no visible surface drying being observed, similar to the behaviorof pure RMF resins. The corresponding aerogels, obtained after supercriticaldrying, varied greatly in physical appearance (nom SiO2), theaerogels retain the typical optical translucency associated with low-densitysilica aerogels, indicating no significant widening of the microstructurenor the presence of large particles that would lead to optical diffraction.The red color of the 90%nom SiO2 aerogel isstrikingly different than that of the pure silica aerogel and alsomore intense than for the aerogels with a higher RMF content, despiteits very low RMF content . We hypothesize that thestrong coloration results from the high color strength of RMF comparedto the nearly completely colorless silica, and the long optical pathdue to the high transparency compared to aerogels with a higher RMFcontent. As the nominal RMF content increases, the aerogels becomeincreasingly less translucent (75 to 63%nom SiO2) and then opaque—thus indicating increased light scattering,pointing toward the presence of larger microstructural features (poresor solid particles). The gradual shift in coloration, regardless ofthe degree of light scattering, is more indicative of changes of thechemical identity.The followingobservations were made for the wet gels: samples with at least 31%pearance 1 and han2 residue, thus allowingfor the determination of the true SiO2 content  under our measurement conditions (up to 800 °C in air),leaving behind a stoichiometric SiO content 2. In add content 2b. For cTo better constrain the chemical composition of the aerogels, C,H, and N concentrations were determined using elemental analysis.The oxygen composition in the organic part was estimated using thefollowing formulaThe calculation was made with the silica content originatingfromTGA results and C, H, and N contents from elemental analysis, andthe remaining mass was attributed to oxygen atoms that were not boundto silica—originating from the RMF structure and silica-boundethoxy and hydroxyl surface groups 3.2 (M = 60.08g·mol–1) and R + M + F (M =57.46 g·mol–1), the molecular (%nom) and weight  nominal composition is nearly equivalent.For compositions >90%nom SiO2, the measuredsilica content is lower than the nominal one. This is simply due tothe presence of the surface groups on the large specific surface ofthe silica aerogel (ethoxy and hydroxyl). If these surface groupswould remain unchanged upon the addition of RMF, which does not seemto be the case based on the observed changes in the ethoxy weightloss peak, one could thus expect a systematic underestimation of theorganic content in the calculated nominal composition. If, on theother hand, the surface groups are partially or fully replaced bythe RMF reactants, this will lead to an increased RMF content withoutchanging the measured silica content. There are also factors leadingto an overestimation of the silica composition. Namely, for the composites,we attributed all remaining mass after TGA up to 800 °C to silica.However, the pure RMF also showed a residue of 5%wt whichclearly cannot be attributed to any specific phase. Thus, the SiO2 content will be slightly over- and the oxygen content underestimatedfor the composite samples. Another factor leading to a larger nominalsilica content is the water formed during condensation of R + M +F species into an RMF resin, lowering the weight of the resin comparedto the reactants. Indeed, for composites with ≤75%nom SiO2, the measured silica content is higher than thenominal one. Although the factors discussed above certainly contributeto this difference between nominal and measured compositions, thereare two factors which indicate that at least for samples with 75 and52%nom SiO2, the RMF yield is less than 100%,and some of the RMF reactants get partially washed away during solventexchange: first, for the 75%nom SiO2 sample,no significant nitrogen concentration is observed, which is contraryto expectations based on the nominal composition. Second, for compositionsbetween 63 and 52%nom SiO2, the measured silicacontent is almost constant or even increases with the decreasing nominalsilica content. This is consistent with observations of particlesin the exchange liquid (see the Supporting Information) and could arise from different degrees of integration for the RMFreagents in the materials’ backbone.1H–29Si and 1H–13C NMR and FTIR spectrawere acquired. All 1H–29Si NMR spectra. Because of the cross polarization during the spectral acquisition,the peak intensities do not directly correspond to the Qn abundances and can thus not be used directly forquantitative analysis: due to its inherently larger distance to 1H, Q4 abundance would be underestimated comparedto Q3 and Q2. The position of the Q3 resonance gives further insights into changes in the bonding environmentas it is sensitive to the nature of the non-bridging oxygen: Q3-OH has an expected peak position around 100 ppm, whereasQ3-O–C typically has a resonance around 104 ppm,regardless of the bonding environment of the carbon.59 The pure silica aerogel  has a relatively broad Q3 peak near 102 ppm indicatinga mixture of silanol and ethoxy groups on the non-bridging oxygen.Upon the addition of small amounts of RMF , the Q3 peak shifts to more negative ppm values, indicating additional organicgroups (−Si–O–C−) chemically bound tothe silica. Further addition of RMF, on the other hand (≤63%nom SiO2), leads to a shift in the opposite directiontoward less negative ppm values, indicating more silanol surface groupsthan in the pure silica. A possible exception is the sample with 52%nom SiO2, where the peak seems to be similar tothe one of the pure silica. However, this is also the sample withthe largest discrepancy between the nominal and measured silica content.According to the measured silica content, the sample should be in-betweenthose with 75 and 63%nom SiO2, which would makeit consistent with the trend described above.To get further insightsin the evolution of the bonding environment,  spectra4a, excepnom SiO2), a significant amountof the additional organic groups seems to react with the surface ofthe silica phase. At higher RMF concentrations, the degree of interactionseems to increase, and even more Si–OH are observed than inpure silica. There are different possible explanations for this. Theorganic groups could attach themselves to the silica surface at anearly stage of the gelation process, partially replacing ethoxy groupsfrom the surface. At higher RMF concentrations, however, this stepcould be followed by a detachment and replacement of the originallyattached organic groups by hydroxyl groups. This could, for example,be driven by the nucleation and growth of a separate resin phase (seeTEM data below). Alternatively, the increase in Si–OH at higherRMF contents could simply be related to a shift in the hydrolysis–alcoholysisequilibrium due to the higher water content in the RMF sol—however,in this case, we would expect a more gradual and continuous changewith an increasing nominal RMF content.The reasons for the observed changes are likely different gelationand integration mechanisms of the RMF phase. At a low RMF content. This means that even for the 31%nom SiO2 sample, which has a measured silica content of only 42%wt, the spectra seem to be dominated by the SiO2-related peaks. Neither of the methods can be used for quantitativeanalysis; NMR due to the cross-polarization discussed for the 1H–29Si spectra and FTIR due to the ATR-basedacquisition conditions and due to the high variability in molar extinctioncoefficients. However, for both techniques, the spectra change graduallyfrom a typical silica spectrum to the one of RMF with a decreasingmeasured silica content. Interestingly though, the signal coming fromthe ethoxy groups does not vanish for either NMR or FTIR, even forthe pure RMF. The 1H–13C NMR of the pureRMF aerogel  which can again be attributedto unhydrolyzed ethoxy groups and an increased intensity in the 1100cm–1 region. The assumption of the presence of ethoxygroups in the RMF phase is based on a similar mechanism describedelsewhere for other alcoholic systems with aromatic molecules, includingmelamine60 and hydroxymethylfurfural,61 similar to the esterification mechanism alsoobserved in silica, with the replacement of terminal O–H groupsby surrounding R-OH alcohols.62 This meansthat care has to be taken not to restrict the interpretation of thepresence of ethoxy groups as forcibly being part of the silica surface.The  aerogel 4b, althonom SiO2, nom SiO2, via TEM, the RMF particles in the 52 and 31%nom SiO2 systems seem to be well integrated in the silicaphase but isolated from each other.TEM imaging shows the expected colloidalnature and random aggregation behavior for both the pure silica andthe pure RMF samples; however, the size of primary particles formingthe agglomerates is very different for silica , we can use the measured properties of thepure RMF. For silica, the situation is more complex, as even the 100%nom SiO2 aerogel contains a significant amount oforganics due to the presence of surface groups. However, for any compositewith a silica content of In the absence of specific interactions and effects, the envelopeand skeletal densities of a physical mixture resemble the volumetricaverage between its components. To be able to estimate the propertiesof such a mixture, we need to know the envelope and skeletal densityof the pure RMF and SiOwt SiO2, the skeletal density of the pure silica phase wasestimated to be 1.77 ± 0.01 g/cm3. To estimate theenvelope density of the pure silica phase, a slightly different approachwas used. The disparity between the porosity in the organic and inthe silica phase being very large, we can neglect any contributionof the organic phase to the porosity in the 100%nom SiO2. Thus, the envelope density of the silica phase can be estimatedfrom the results of the 100%nom SiO2 sample,using the total pore volume to solid volume ratio, that is, porosity, On applyingthis formula to the two samples with >85%With the definitions of pore volume, XwtSi) using Once both the skeletal and envelope densities of the pure phases are known, the expected total densitiescan be calculated from the weight percent of silica .To get further insights into this possible densification, the porosityof the samples has been assessed using nitrogen sorption analysis,which probes the porosity in the scale of ∼1–50 nm.Nitrogen physisorption usually results in a calculation of the surfacearea/pore volume per gram of material. Although this assessment isadapted to single-component systems, composites have different skeletaldensities: a given volume of an RMF solid is lighter than the samevolume of amorphous silica, leading to a large volumetric variationin what constitutes a gram of solid material. Therefore, surface areasand mesopore volumes are instead discussed in, respectively, mS of the samples gradually decreasesfrom typical values for silica aerogels (1428 m2/cm3 or 835 m2/g for SNLDFT and 1573 m2/cm3 or 920 m2/g for SBET) to typical values for RMF resins (77 m2/cm3 or 67 m2/g SNLDFT and 100 m2/cm3 or 51 m2/g SBET) with a decreasing silica content(see the Supporting Information for thefull data set). To understand a possible densification, we comparedboth the measured NLDFT pore to solid volume ratio (Vpore/solNLDFT) and the total pore to solid volume (Vpore/soltot) to theexpected trends Vpore/solorg). The results (d = Vpore/solNLDFT/SNLDFT) has been calculated , organic species from the RMF precursors seem to partially replacethe hydroxyl and ethoxy groups from the silica surface, leading toa shift in the ethoxy peak observed in TGA and a shift of the Q3 NMR peak indicating more carbon-rich environments. The additionalorganic surface groups seem to contain little to no nitrogen, indicatingthat melamine might contribute less to their formation and is likelyto be washed out partially during solvent exchange. The interactionbetween the RMF precursors and silica surfaces, together with thelower initial RMF concentration in the sol, seems to prevent the formationof a separate RMF-resin phase: no such phase can be observed in TEM.It is likely that an initial adsorption of RMF precursors on the silicasurfaces present in the sol happens even in systems with a lower silicacontent. However, for systems ≤63%nom SiO2, RMF particles do nucleate and grow as can be seen from the TEMimages. However, both the dilution of the RMF with the addition ofthe silica sol and the interaction between RMF precursors and silicasurfaces lead to a lower super-saturation with respect to the resincompared to the pure sol. This results in much larger primary RMFparticles and will likely also slow down the nucleation and growthkinetics. At least for systems between 63 and 52%nom SiO2, the formed RMF particles seem to be only partially integratedin the aerogel, either due to their size leading to precipitation/segregationor due to the slow nucleation/growth kinetics. With even a lower silicacontent, the RMF particles seem to be well integrated in the silicaphase. The formation of RMF particles seems to lead to the at leastpartial desorption of precursor species from the silica surface andtheir integration into the RMF phase. These surface groups seem tobe replaced by hydroxyls, leading to another shift of the NMR Q3 peak.Together with the chemical identity assessment, the microstructuralanalysis allows us to formulate a hypothesis with some confidenceon what happens in these composite systems during gelation. At highnominal silica concentrations . Some samples displayed clear signs of macroscopicdefects during the measurements—these measurements were notused for further analysis. For the remaining measurements, three differentquantities were determined: the minimum of the modulus in the viscoelasticplateau, the maximum stress reached and the strain at maximum stress,and the stress and strain at the first clearly discernible localizedfailure. Although the definition of the first two is clear, the firstlocalized failure is less clear. We used two criteria for the definition:the modulus needed to drop locally below zero, and a discontinuityof the stress–strain curve needed to be visible.The mechanicalcompression behaviorof aerogels is complex and not fully understood. For our composites,similar to previous observations on silica aerogels,2 sample, the sampleswith 75%nom SiO2 (with organic groups at thesilica surfaces) seem to show a moderate improvement of the mechanicalproperties. Although the minimum modulus of the viscoelastic plateauis in the lower range of what has been measured for the pure system,the samples support both a higher stress and a higher strain beforeboth localized and global failures. For lower amounts of silica, resultingin samples with RMF particles embedded within the silica phase, nosignificant improvement of the mechanical properties can be found.At 52%nom SiO2, resulting in a sample with asimilar measured SiO2 content as the 75%nom SiO2, the minimal modulus is lower than the one of the pure silicasample, and while a similar stress is reached before failure, theresulting strain is larger indicating an overall lower modulus. Finally,samples with the least amount of silica (10%nom SiO2) are overall significantly weaker, failure occurs at lowerstresses, and a lower minimal modulus than that of pure silica aerogelis observed. This is likely a result of the lack of connectivity ofthe RMF phase. Indeed, the incorporation of large RMF particles whichare much less compressible than those of the silica phase is likelyto lead to strain concentrations in the silica phase. Additionally,the mechanical property of the silica–RMF interface is notwell known and could contribute to a weakening of the composites.In conclusion, while the adsorption of additional groups at the surfaceof the silica seems to lead to a moderate improvement of the mechanicalproperties, the inclusion of large, isolated RMF particles in thesilicate phase, while having little effect on the thermal conductivity(see below), does not improve the mechanical properties, and dependingon the concentration can even have a detrimental effect.Thecompression of silica aerogels reveals various behaviors dependenton the actual silica concentration and the microstructural distributionof the two phases 8. Comparnom SiO2 sample.To estimate the performance ofthe composites for different applications, their thermal conductivityand mechanical compression behavior was measured. For the thermalconductivity, we again compare to trends for mechanical mixtures.For this, we estimated the two limiting cases of purely parallel andpurely serial resistances from the two phases. As for the densities,we first needed to estimate the thermal conductivity of the pure silicatephase in the serial 7 and parOnce the conductivity of thepure silicaphase is calculated, we can estimate the expected thermal conductivitiesfor the serial and parallel case with nom SiO2) displays variations that are all withinthe measurement accuracy, allowing no conclusions with respect todifferences in behavior. For an intermediate silica content, (10–31%nom SiO2) thermal conductivity clearly follows thetrend of serial resistances, while for a lower silica content (5%nom SiO2), the measured thermal conductivity iscloser to that of the parallel case. This is consistent with the observedlack of interconnectivity between the RMF particles down to silicacontents as low as 10%nom SiO2. The measuredthermal conductivity of the 5%nom SiO2 sample,on the other hand, indicates a continuous RMF network having formedthroughout the aerogel volume. These results indicate a thresholdfor high insulation performance somewhere between 5 and 10%nom SiO2, corresponding to 14–24%wt and50–70%vol of SiO2, this threshold beingdue to a progressive connectivity of RMF particles happening throughoutthe material.The resultsof the thermal conductivity are shown in nom SiO2), the organic phase gets incorporated as additionalsurface groups or even a surface coating of the silica phase. Contraryto the precursors, this surface coating contains little to no nitrogen.At lower silica contents, a separate RMF resin phase forms. For silicacontents ≃75%nom SiO2, the RMF phaseis integrated as large (200–400 nm), isolated particles withinthe silica phase. Either due to the interaction of the RMF precursorswith the silica phase or due to dilution, the size of these particlesis significantly larger than that of the primary particles in thepure RMF resins. At intermediate silica levels (52–63%nom SiO2), the RMF particles are only partiallyintegrated in the aerogels, leading to final compositions that differfrom their (expected) nominal composition. At silica contents ≤5%nom SiO2, a connectivity of the RMF phase is reached,significantly changing the thermal conductivity which, up to thispoint, remains similar to that of pure silica aerogels. Although theaddition of an organic surface coating leads to a moderate improvementof the mechanical properties, the isolated RMF particles have no sucheffect and, depending on the concentration, can even be detrimental.With their tailorable microstructure and very low thermal conductivity,RMF–silica composite aerogels have potential applications forthermal insulation or as green bodies for the production of silica–carboncomposite aerogels.In this work, we have taken advantage of thecompatibility of twosol–gel systems—TEOS-based silica and RMF alcogels—toproduce RMF–silica hybrid aerogels by supercritical dryingof the alcogels. Thorough chemical and microstructural analysis andmeasurement of the resulting properties have provided an in-depthunderstanding of the gelation process and resulting microstructurewithin the composites. At a high silica content (≥75%


The irrigation every 3 d was considered as the control in the current study. For this purpose, biochar was prepared through the pyrolysis of corn stalk and rice husk at 350 °C for 3 h, while sonochemical combined with the precipitation method was used to prepare zinc oxide nanoparticles (ZnO NPs) from zinc acetate. The morphological structures of the produced biochar and ZnO NPs were characterized using X-ray diffraction (XRD), N2 gas adsorption-desorption, scanning electron microscopy (SEM), and transmission electron microscopy (TEM). The results exhibited that the combination of biochar alongside ZnO NPs resulted in a positive significant effect on the physiological traits such as chlorophyll content, relative water content, plant height, and leaf area index as well as yield-associated components , and biological and grain yield ha−1 when rice plants were irrigated every 9 days without a significant difference with those obtained from the control treatment (irrigation every 3 d). In conclusion, the combination of biochar and ZnO NPs could be recommended as an optimal approach to maximize both grain yield ha−1 and WUE of rice.Water stress is considered one of the most environmental hazards that threaten agricultural productivity. Therefore, two field experiments were conducted to investigate the impact of biochar (6 t ha Oryza sativa L.) as a cereal crop is an important food source for the most of the world’s population  at the rate of 5 L/ha at 5 days after transplant. Nitrogen in the form of urea (46% N) at the rate of 165 kg N ha−1 was applied as recommended in two batches, 66.67% as a basal application and 33.33% at panicle initiation. Calcium superphosphate (15% P2O5) and potassium sulfate (48% K2O) were applied at a recommended dose at a rate of 37 kg P2O5/ha and 50 kg K2O/ha, respectively.The prior cultivated crop was wheat during both seasons of the study. Pre-germinated healthy rice seeds (2 (6 t/ha) as soil amendment, and their combination between the exogenous application of ZnO NPs and biochar application (BC + ZnO NPs). The BC was distributed and mixed with soil  homogeneously in treated plots. However, treatments of ZnO NPs had been applied at two physiological stages of rice .The permanent cultivable location of the experiment was well plowed (plowing two perpendicular times) and leveled. The experiment was laid out as a split-plot design with four replicates. The main plots were devoted to the four irrigation deficit treatments; irrigation every 3 (control), 6, 9, and 12 days which are denoted as ID3, ID6, ID9, and ID12, respectively. Whilst, the subplots were occupied by the four treatments of control, exogenous application of ZnO NPs at 50 mg/L, biochar (BC) at 0.6 kg/mChlorophyll content in flag leaves was measured using a SPAD meter device . Leaf area index (LAI) was estimated in the heading stage as described by  accordinRelative water content (RWC) was estimated according to  as givenFW: the weight of healthy and fresh leaves.TW: the watery saturated weight after rehydrating leaves in distilled water for 24 h.DW: the dry weight of water-saturated leaves after drying procedure at 80 °C for 24 h in an oven.2. Ten panicles were randomly collected to determine the total filled grains number/panicle, unfilled grains number/panicle, and 1000-grain weight (g). Biological yield and grain yields per ha were measured from an area of 12 m2 (3 × 4 m) which was harvested from the inner of each plot to avoid the border effects. Grain yield was adjusted to 14% moisture content. Harvest index (HI) was calculated as the ratio of economic yield  to biological yield  according to [Reproductive tillers of five hills were counted from the midmost of each plot at maturity to estimate the number of panicles/mrding to .Experiment irrigation has been conducted using calibrated water meter with a water pump to estimate the amount of applied water according to each treatment. Water use efficacy (WUE) was calculated by the following formula:The obtained data from the effects of biochar, ZnO NPs and their combinations treatments on growth, physiology and productivity of rice grown under irrigation deficit were subjected to analysis of variance according to . The meaSEM was used to clarify the surface morphology of the as-synthesized biochar, since such parameters are important for biochar characteristics that can affect the soil and plants in next sections. Taking into account, the scanning was carried out at three different magnifications to observe all presented features formed with biochar. It can be observed from X-ray diffraction was used to determine the structural characteristics of ZnO NPs. 2/g. As shown in the inset figure, the pore volume and mean pore diameter were 0.063 cm3/g and 2.3 nm, respectively. SEM imaging was used to analyze the shape of ZnO NPs. As shown in The physiological traits are very important to be measured to show the negative impacts of water stress or the mitigation role of BC and ZnO NPs in plants grown under irrigation deficit in further improving water productivity and drought adaptation is infinite, since such traits are considered the real indicator or the plant health and productivity in different stages. Data presented in In addition, there was not a statistical difference between ID3 and ID6 in all physiological studied traits in both cultivated seasons except for the RWC characteristic where a moral statistic difference between them occurred in the 2019 season. As for the applications of ZnO NPs, BC, and BC + ZnO NPs, it is clear that these treatments have positively improved on all physiological traits studied . The bes2 (by 44.1 and 43.3%), and filled per panicle (by 43.2 and 44.1%) in 1st and 2nd seasons, respectively ; both number of panicles/mectively . Inverse2 underlying both ID3 × BC + ZnO NPs and ID6 × BC + ZnO NPs treatments without significant difference among them in the 2019 and 2020 seasons. Whilst, the least panicles number per m2 is located under the interactive treatments of ID12× control in both seasons. Concerning the number of filled grains/panicle, the same trend was displayed in 2019, whereas the combination of ID3 × BC + ZnO NPs and ID6 × BC + ZnO NPs rendered the choicest number of filled grains/panicle in comparison to all interactive treatments , yield reduction (%), water saved (%), and water use efficacy (kg/m3) in 2019 and 2020 seasons. It is obvious that ID3 represented the highest value of aggregate water applied (m3/ha), whilst the least amount of water applied was recorded under ID12 in both cropping seasons. At the same time, ID3 treatment occupied the lowest percentage of both water saving and grain yield reduction. However, ID12 treatment rendered the highest percentage of both water saving and grain yield reduction in 1st and 2nd seasons. Interestingly, in each season, prolonged irrigation intervals until 9 days (ID9) had the maximum value of water use efficacy. The irrigation treatment every 6 days (ID6) gave the second-best efficacy of water use in both rice seasons. Therefore, irrigation treatment every 9 days could be an ideal application in this study.Data listed in Concerning the combination effect of irrigation deficit and applications of BC, ZnO NPs, and BC + ZnO NPs treatments on water use efficacy, appraisal results pointed to the superiority of ID9 × BC + ZnO NPs in giving the most efficient use of water in both seasons followed by ID6 × BC + ZnO NPs in 2019 and 2020 seasons . HoweverBenign growth traits are conclusive proof of a healthy plant environment. Additionally, the plant’s chlorophyll content is evidence of photosynthetic efficacy because it acts as a transfer and harvester of light energy . Indeed,+-N, NO3-N, and K nutrient concentrations in the soil when irrigation treatment was every 6 and 9 days. These results were well supported by the research studies conducted by [Our findings deduced that the rice crop does not need large amounts of water to produce a high yield outcome if it has adequate and suitable water addition. Therefore, there wasn’t a significant difference between ID3 and ID6 concerning final grain output . Probablucted by ,38,39.3 plants and this is considered a good contributor for enhancing the efficacy of gas exchange underlying harsh conditions [In the current study, the application of ZnO NPs as exogenous treatment improved the growth, physiological, yield and water use efficiency of rice grown under water stress treatments. Separately, the application of ZnO NPs protects the leaf surface from harmful sun rays by blocking UV radiation . Probablnditions . Furthernditions .In the current study, the unique synthesis of ZnO NPs (a uniform size with average particles ≤ 50 nm) via the chemical precipitation method  might reOn the other hand, the controlled pyrolysis method of feedstocks material under limited oxygen conditions can lead to a sequester riches carbon materials . BiocharBiochar properties can improve soil fertility and moisture-holding capacity, which can result in an amending for water requirements with maintaining benign yield production. Moreover, the sole application of BC enhances the water holding capacity of cultivated soil reflected positively in producing better water availability and improving the efficacy of water use feature ,10. The As per our findings during the current investigation, the combined application of BC and ZnO NPs resulted in a significant improvement into the physiological, yield components, and grain quantity traits compared to the individual application of either BC or ZnO NPs. This can be possibly due to the accompaniment of BC as soil amendment and ZnO NPs as exogenous applications which can enhance different nutrients acquisition and water enabling in roots zone as well as the amelioration effects of ZnO NPs on plant growth grown under irrigation deficit conditions. On this basis, the combined effect of BC as soil amendment and ZnO-NPs as exogenous applications can positively improve water relations and enhance crop yield production under irrigation deficit treatments.−2, number of filled grains per panicle, biological yield, grain yield and 1000-grain weight, while the lowest values of those traits were obtained when plants irrigated each 9 days. Under a severe water deficit, the influence would be devastating on the plant without the interference of agronomic treatments that would alleviate this negative effect. Accordingly, biochar preparation through the pyrolysis of the corn stalk and the rice husk (wastes) showed flake-like morphology with varied sizes in micrometer ranges and the formation of hollow structures that are extended to micrometer sizes. In addition, the sonochemical precipitation of synthesized ZnO NPs gave homogeneous nanoparticles with a hexagonal phase and crystalline size of 48.70 nm, a specific surface area of 54.26 m2/g, pore-volume, and mean pore diameter of 0.063 cm3/g and 2.3 nm, respectively. Though, Amendment of paddy soils with BC which is carbon-rich material has been proposed as a safe approach to abate water deficit cases via enhanced water availability in root growth zone environments. On the other hand, the application of ZnO NPs is considered unique stimulants nutrients that mitigate the undesired influence of water stress to an appreciable extent. The application of BC alongside ZnO NPs foliar sprayed has a synergistic impact on the enhancement of the physiological traits and yield-related attributes, and water use efficacy compared with the sole application. In the paddy cultivation system, more applied research is needed to validate these obtained findings under diverse varieties, soil types, different stressors as well as various climatic conditions.Water irrigation deficit has a cardinal action in determining the final grain production, which grossly affected plant physiological traits, yield constituents, and consequently grain yield productivity. In our investigation, irrigation of rice plants each 3 or 6 days resulted in the highest values, without significant differences, of plant height, number of panicles m


Macromolecular proton fraction values proved to be reduced  in the patient's cerebral peduncles by 3% and in the pons by 4.1% and in a periventricular white matter lesion by 6.4% . Based on our findings, we argue that quantitative MRI techniques may be a valuable source of biomarkers and reliable diagnostic criteria and can shed light on the pathogenesis and exact nosological position of this disorder.Chronic lymphocytic inflammation with pontine perivascular enhancement responsive to steroids (CLIPPERS) is a rare chronic central-nervous-system inflammatory disorder that became known only recently, and the pathogenesis of CLIPPERS remains poorly understood. This report presents clinical and radiological features of a rare case: a young female patient who rapidly died of suspected CLIPPERS. Helpful multiparametric MRI diagnostic criteria are proposed that can help discriminate CLIPPERS from non-CLIPPERS pathologies. We reviewed clinical history, symptoms, quantitative data from brain multiparametric MRI before and after treatment, and histopathological data. Perfusion-weighted imaging revealed a decrease in regional cerebral blood flow by 31% and in cerebral blood volume by 64%, with a moderate increase in transit time and in time to peak by up to 23% in affected pontine and cerebral white matter. As estimated by diffusion tensor imaging, there was elevated density of tracts (n/mm Chronic lymphocytic inflammation with pontine perivascular enhancement responsive to steroids (CLIPPERS) is a rare chronic central nervous system (CNS) inflammatory disorder that became known only recently  In this case report, clinical and radiological features of a young female patient are presented (who rapidly died of suspected CLIPPERS) to propose helpful multiparametric MRI diagnostic criteria to discriminate CLIPPERS from non-CLIPPERS pathologies. We also aimed to review the clinical history, symptoms, quantitative data from brain multiparametric MRI before and after treatment, and neuropathological-examination results.A 33-year-old woman presented with episodes of a recurrent visual disturbance, mostly in the form of unilateral visual loss, refractory and severe headache, and aversion to some foods . The patient's symptoms started in November 2021, 2 days after an unremarkable brain injury. She had no relevant medical history. Family medical history revealed that her aunt has multiple sclerosis (MS). Her initial brain MRI revealed multiple areas of curvilinear and punctate enhancement in the pons and other specific features described above, which made the diagnosis of CLIPPERS more probable  and 2. AOn 10 December 2021, the patient was hospitalized at the State Novosibirsk Regional Clinic, Neurologic department. A neurological examination showed increased tendon reflexes S>D; the finger-to-nose test and heel-to-shin tests revealed dysmetria and intention tremor, stronger on the left side. An ophthalmological examination indicated bilateral optic neuritis. Blood biochemical tests revealed elevated levels of urea and creatinine: 184.9 and 21.9 mmol/L, respectively. Consistently with persistently high blood pressure including periodic sudden spikes up to 260/180 mm Hg, a diagnosis of probable chronic glomerulonephritis was made.Other than that, in all laboratory and serological tests, no substantial and specific aberrations were detected. A complete blood count, results of cerebral spine fluid (CSF) biochemical tests, the C-reactive protein level, and the erythrocyte sedimentation rate were unremarkable. Tests for infectious agents  yielded no findings that would point to an infection. Immunological tests detected no autoantibodies and markers of vasculitis. Onconeuronal antibodies  in serum and oligoclonal bands in CSF were not detectable either.After stabilization of blood pressure, she was prescribed dexamethasone 24, 16, and 8 mg per day for 3 days each dose. A second MRI was performed in the Neurological Hospital on 27 December 27, 2021. Unfortunately, no additional quantitative research methods were applied in the scan protocol there. Nonetheless, judging by an almost 100% reduction in T2 and a fluid attenuated inversion recovery (FLAIR) strong signal and pontine enlargement, a 3-fold reduction in the apparent diffusion coefficient in the most affected pons tissue, she experienced a really promising and rapid radiological improvement .According to almost complete radiologic resolution and clinical recovery, the patient was discharged from the hospital and was kept under observation as an outpatient starting on December 27. The patient continued to take methylprednisolone orally by herself in January 2022 at a dose of 24 mg a day. On the 21st of January, the day before a planned control multiparametric MRI, the patient suddenly died at home without any witnesses.Neuropathological analysis revealed no remarkable macroscopic changes in the brain, except for an uneven pattern of cerebral edema. A forensic histological brain examination dated February 3, 2022, yielded some nonspecific microscopic findings such as contoured neurons, swollen basophilic nuclei, glia proliferation, dilated perivascular and pericellular spaces, and a thickening of capillary and artery walls. Even without proof from the neuropathological analysis and laboratory tests, a diagnosis of probable CLIPPERS seemed to be the most appropriate explanation during the patient's single flareup, as evidenced by the recovery caused by the clinical corticosteroid therapy, imaging characteristics on pre- and post-treatment MRI, and the subsequent death.The 33-year-old woman underwent brain MRI in the Medical Department of the International Tomography Center  because of visual disturbances and headache suggestive of neuritis. She had no relevant problems in her medical history. Images were acquired using a 3-T Scanner  with the manufacturer's transmit-receive head coil. Besides the standard structural brain MRI protocol, including T1 turbo field echo 3D and T2-weighted image (WI), we employed FLAIR 3-dimensional (3D), susceptibility-weighted imaging, diffusion-weighted imaging (DWI), 3D time-of-flight angio, post-Gad-T1 turbo field echo 3D, research sequences diffusion tensor imaging (DTI), T1 perfusion, and a fast 3D whole-brain macromolecular proton fraction (MPF) mapping protocol. The neuroimaging data were interpreted by 3 neurologists as probable CLIPPERS by consensus.MRI of the brain uncovered disseminated and mostly symmetrical regions of distributed infiltrative aberrations localized bilaterally to the pons and to adjacent hindbrain structures  with similar multifocal spots in periventricular cerebral white matter manifesting T2/FLAIR signal hyperintensity -C, E, F.There was no radiological evidence of a vascular pathology such as vasculitis or vascular lymphoma on the intra-extracranial MR-angiogram . UnremarAdditional perfusion-weighted imaging (PWI) was performed in the dynamic susceptibility contrast mode: T2*-weighted 3D gradient echo planar imaging sequence , in the axial plane . To determine quantitative characteristics of PWI, we delineated a region of interest of round and irregular shape in the lesions, followed by automatic transfer of the geometrical template to all synchronized images. PWI-derived regional cerebral blood flow (CBF)-mapped images revealed a “blue” pattern within pons peduncles and a cerebral white matter lesion A, D, conGraphs of the “signal intensity – time” dependence in each pixel of the slice revealed a decrease in regional CBF by 31% and in cerebral blood volume by 64% with a moderate increase in transit time and time to peak up to 23% in affected pontine and cerebral white matter when compared with unaffected white matter 2) and diffusion parameters (×10−3 mm/s2) in the patient's pons as compared to the healthy control were as follows: DOT = 13.5 vs 12.4, mean diffusivity = 1.05 vs 0.87, axial diffusivity fractional anisotropy = 1.42 vs 1.29, radial diffusivity = 0.87 vs 0.66, and fractional anisotropy = 0.32 vs 0.45. Considering the visual and quantitative results of DTI, the obtained data can be interpreted as an increase in interaxonal distance with intercellular edema and/or thickening of the inflamed myelin sheath.Visual representation of corticospinal tract fibers in the region of the affected hindbrain on initial DTI tractography showed slightly asymmetrical irregular fibers with preserved density A. In theTo evaluate the degree of secondary myelin loss, we carried out a new test—fast whole-brain 3D MPF mapping—well proven as a sensitive assay of myelin by several recent animal and human studies . We recoWhite matter MPF was found to be reduced  in cerebral peduncles by 3% and in the pons by 4.1%; in the periventricular white matter lesion, MPF proved to be reduced by 6.4%  .A side benefit of the 3D-MPF mapping is the ability to perform white matter/gray matter/CSF segmentation and volume (V) calculation, which can help to evaluate lesion volume comparable data for precise assessment of dynamics of a process after treatment. According to extremely low MPF mean values in the lesion (3%- 6.4%), we propose that it belongs to the automatically generated gray matter class, and therefore the indicative lesion volume can be roughly estimated as a white matter V reduction.3 (MPF 10.2%) as compared to the control 495.8 cm3 (MPF 11%). Volume of gray matter with demyelinated lesions was 748 cm3 (MPF 5.5%) as compared to the control: 737.8 cm3 (MPF 5.8%). CSF volume was 251.2 cm3 compared to the control 330.3 cm3. Calculated lesion volume was ∼10.5 cm3 with an intact white matter MPF reduction of 7.2%.In our patient, intact white matter volume was found to be reduced to 485 cmA second post-treatment MRI on December 27 performed at Novosibirsk State Regional Clinic showed almost complete radiologic resolution, no residual enhancement, no high signal on DWI, and no additional brain anomalies .Fig. 6ReIn this report, we present a unique fatal case of a briefly observed clinical history of a patient as well as repeated MRI with several atypical features fulfilling revised criteria for CLIPPERS According to the distinct MRI features supported by multiparametric quantitative MRI data , well-pronounced clinical and radiological improvements induced by corticosteroid therapy, followed by rapid clinical worsening fatal to the patient, the most suitable diagnosis is CLIPPERS It has been proposed that a relapse with atypical symptoms or radiological features should be a “red flag” suggestive of a diagnosis of CLIPPERS In terms of PWI, our results are consistent with another study 3 as compared with that in a healthy volunteer: 12.5 n/mm3. At the same time, the indicators of average, axial, and radial diffusion also turned out to be increased, and fractional anisotropy was found to be decreased accordingly. Above the lesion under study, DTI quantitative data on specific alterations can be interpreted as a denser but less organized distribution of nerve fibers, related to an additional (inflammatory) factor in the intercellular space. Our PWI and DTI results are in agreement with known CLIPPERS biopsy features, namely predominantly T-cell infiltration in hindbrain white matter, largely perivascular in terms of distribution, and accompanied by a moderate number of histiocytes and activated microglia [According to our DTI data, the DOT in the patient's area of interest (pons) was elevated to 13.5 n/mmicroglia ,15.Dramatically lowered MPF mean values in lesions (2-5 times less than those in controls) indicate a remarkable myelin loss and highlight added diagnostic value of MPF mapping for ruling out subtentorial tumors containing a substantial collagen compartment because such tumors usually have high MPF values Simon and coworkers have also pointed out that varying degrees of myelin loss in small areas may accompany a highly intense inflammatory infiltrate (regarded as a secondary phenomenon) and focal neuronophagia, which has been observed in some cases ,11. MoreAlthough ideally, neuropathological analysis should be performed in suspected CLIPPERS, this approach will be infeasible in many cases where vital CNS regions are affected primarily, as in our case. A blood or CSF sample of the patient during and after a flareup is not always available for antibody tests to exclude other autoimmune CLIPPERS-like diseases that should be taken into account in the differential diagnosis: autoimmune glial fibrillary acidic protein astrocytopathy Accordingly, we propose to consider potential usefulness of the multiparametric and quantitative MRI radiologic features presented in our case. It was demonstrated here that these radiologic MRI features are of paramount importance for a preliminary diagnosis and for determining the severity of CLIPPERS even in the case when the only symptom  in combination with young age did not help to discriminate CLIPPERS from non-CLIPPERS, especially from MS or opticomyelitis. MRI with a whole spectrum of structural and quantitative tools remains a crucial source of diagnostic criteria for CLIPPERS that are necessary to ensure that alternative diagnoses are not overlooked and that treatment will be successful.Diagnosis of CLIPPERS is challenging and requires careful exclusion of alternative diagnoses. Typical and severe radiological features lacking clinical manifestations, marked corticosteroid responsiveness, and worsening after corticosteroid withdrawal are demonstrated in our case as hallmark features of CLIPPERS; we propose them as signs of “probable CLIPPERS.”  Because a specific serum or CSF biomarker of the disorder is currently unknown and neuropathological criteria are lacking, we suggest adding multiparametric and quantitative MRI analyses as promising techniques for intravital verification of the diagnosis; they include PWI, DTI, and MPF mapping in scan mode when CLIPPERS is suspected. We would like to emphasize that physicians should be aware of this condition and relevant differential diagnoses to take advantage of the benefits of the early diagnosis and GCS-based therapy, as in our case. Currently, extensive multimodal investigations, further studies to determine potential usefulness of quantitative MRI findings as biomarkers, and reliable diagnostic criteria are necessary; they can shed light on the pathogenesis and exact nosological position of this disorder, which remains poorly understood.All authors provided substantial contributions to the manuscript content. All authors gave final approval of the version of the article to be published.The experiments involving human subjects were reviewed and approved by the Institutional Review Board of the International Tomography Center (the Siberian Branch of the Russian Academy of Sciences). The patient and her husband provided their written informed consent to participate in this study and for the publication of any potentially identifiable images or data included in this article.Written informed consent for publication of this case report was obtained from the patient. All images were anonymized and contain no personally identifiable information.


The English Bulldog has risen sharply in popularity over the past decade but its distinctive and extreme conformation is linked to several serious health conditions. Using multivariable analysis of anonymised veterinary clinical data from the VetCompass Programme, this study compared the odds of common disorders between English Bulldogs and all remaining dogs in the UK during 2016.From 905,544 dogs under veterinary care during 2016, the analysis included a random sample of 2,662 English Bulldogs and 22,039 dogs that are not English Bulldogs. English Bulldogs had 2.04 times the odds of diagnosis with ≥ 1 disorder than dogs that are not English Bulldogs . At a specific-level of diagnostic precision, English Bulldogs had increased odds of 24/43 (55.8%) disorders. These included: skin fold dermatitis , prolapsed nictitating membrane gland  and mandibular prognathism . Conversely, English Bulldogs had significantly reduced odds of 6/43 (14.0%) disorders. These included: retained deciduous tooth , lipoma  and periodontal disease . At a grouped-level of diagnostic precision, English Bulldogs had significantly increased odds of 17/34 (50.0%) disorders. These included: congenital disorder , tail disorder  and lower respiratory tract disorder . Conversely, English Bulldogs had significantly reduced odds of 3/34 (8.8%) disorders. These were: dental disorder , spinal cord disorder  and appetite disorder .These results suggest that the health of English Bulldogs is substantially lower than dogs that are not English Bulldogs and that many predispositions in the breed are driven by the extreme conformation of these dogs. Consequently, immediate redefinition of the breed towards a moderate conformation is strongly advocated to avoid the UK joining the growing list of countries where breeding of English Bulldogs is banned. The English Bulldog has risen sharply in popularity over the past decade in the UK. However, its distinctive and extreme conformation has raised concerns because many of these physical features render the breed prone to serious health conditions. There are increasing international efforts to either reform the breed towards a more moderate conformation or, in the absence of such change, to ban the breeding of these dogs on welfare grounds.This VetCompass study explored random samples of anonymised veterinary clinical records from 2,662 English Bulldogs and 22,039 dogs that are not English Bulldogs from an overall population of 905,544 dogs under veterinary care during 2016 in the UK to identify all health disorders recorded for each dog during 2016. The most common disorders in each group were identified and the risks for the most common disorders overall was compared between the English Bulldogs and dogs that are not English Bulldogs.English Bulldogs were younger (2.65 years vs 4.42 years) and heavier (25.55kg vs 13.54kg) than dogs that are not English Bulldogs. English Bulldogs had 2.04 times the risk of diagnosis with at least one disorder during 2016 compared with dogs that are not English Bulldogs. English Bulldogs had increased risk for 24/43 (55.8%) disorders compared to dogs that are not English Bulldogs. The disorders with the highest risk in English Bulldogs included skin fold dermatitis (× 38.12), prolapsed nictitating membrane gland [cherry eye] (× 26.79), protruding lower jaw (× 24.32), brachycephalic obstructive airway syndrome [BOAS] (× 19.20) and interdigital cyst (× 12.96). Conversely, English Bulldogs had significantly reduced risk of 6/43 (14.0%) disorders compared to dogs that are not English Bulldogs. These included: retained deciduous [baby] tooth (× 0.02), lipoma [fatty mass] (× 0.06), dental disease (× 0.23), itchy skin (× 0.25) and flea infestation (× 0.40).The results of this study suggest that the overall health of the English Bulldog is much lower than dogs that are not English Bulldogs. Many of the disorder predispositions reported in this study are intrinsically related to the extreme conformation of English Bulldogs. Redefining the body-shape of the breed away from its current extreme conformation and towards a moderate conformation is advocated for urgent action to avoid the UK joining the growing list of countries where breeding of English Bulldogs is banned. The (English) Bulldog was originally a muscular and athletic animal bred and used to attack bulls for sport, broadly similar in conformation to the modern Staffordshire Bull Terrier . After tThis refashioned Bulldog had more exaggerated conformation than its ancestors – in particular, a shorter face with a protruding underjaw, heavier build and bowed forelegs . Becauseth most numerous breed registered by the Kennel Club, comprising 0.5% of all registrations that year [From the start of the twentieth century, veterinary canine specialists freely acknowledged the (English) Bulldog’s predilection to conformation-related disease , 19. As hat year . Yet thehat year , 22. Deshat year .During the last decade, following the recommendations of the Bateson report , there hUsing anonymised veterinary clinical data from the VetCompass Programme , this stThe study population included all available dogs under primary veterinary care at clinics participating in the VetCompass Programme during 2016. Dogs under veterinary care were defined as those with either a) at least one electronic patient record (EPR)  recorded during 2016 or b) at least one EPR recorded during both 2015 and 2017. VetCompass collates anonymised EPR data from primary-care veterinary practices in the UK for epidemiological research . Data fiA cross-sectional study design was used to estimate and compare the one-year (2016) period prevalence of the most commonly diagnosed disorders in a random sample of English Bulldogs and a random sample of all other dogs. Power calculations estimated that 2,184 English Bulldogs and 21,832 dogs that are not English Bulldogs were needed to detect an odds ratio of ≥ 1.5 for a disorder occurring in 2% of dogs that are not English Bulldogs, with 80% power and 95% confidence and assuming a 10:1 ratio of dogs that are not English Bulldogs to English Bulldogs , 34. EthBreed information entered by the participating practices was cleaned and mapped to a VetCompass breed list derived and extended from the VeNom Coding breed list . Dogs reThe list of unique animal identification numbers for all dogs under veterinary care in 2016 was randomly ordered and the clinical records of a randomly selected subset of animals were reviewed manually in detail to extract the most definitive diagnoses recorded for all disorders that existed during 2016 . ElectivOne-year period prevalence values were reported separately for English Bulldogs and dogs that are not English Bulldogs to describe the probability of diagnosis at least once during 2016. The final combined list of common disorders included the 30 most common disorders in English Bulldogs and the 30 most common disorders in dogs that are not English Bulldogs. Categorical data were summarized with number (percent) and continuous variables were summarised using median, interquartile range (IQR) and range. Mann–Whitney U test, chi-square test and Fisher’s exact test were used as appropriate for comparison of demographic data between cases and non-cases . MultivaThe study population of 905,544 dogs under veterinary care during 2016 in the UK included 8,410 English Bulldogs (0.93%) and 897,134 dogs that are not English Bulldogs (99.07%). Random samples of 2,662/8,410 (31.65%) English Bulldogs and 22,039/897,134 (2.46%) dogs that are not English Bulldogs were included in the analysis. Data completeness were breed 99.7%, age 98.8%, sex-neuter status 99.7%, insurance status 100.0% and bodyweight 65.5%.p < 0.001). The median adult bodyweight of English Bulldogs  was heavier than for dogs that are not English Bulldogs  (p < 0.001).Descriptive results were reported on 2,662 English Bulldogs and 22,039 dogs that are not English Bulldogs Table . The medp < 0.001).Of the English Bulldogs, 2,017/2,662 (75.8%) were diagnosed with ≥ 1 disorder during 2016 compared with 14,534/22,039 (65.9%) dogs that are not English Bulldogs. After using multivariable methods to account for effects of age, sex-neuter status, at/above or below mean bodyweight, insurance status and vet group, English Bulldogs had 2.04 times the odds of diagnosis with ≥ 1 disorder than dogs that are not English Bulldogs , prolapsed nictitating membrane gland , mandibular prognathism , BOAS  and interdigital cyst . Conversely, English Bulldogs had significantly reduced odds for 6/43 (14.0%) specific-level disorders compared to dogs that are not English Bulldogs. These included: retained deciduous tooth , lipoma , periodontal disease , pruritus  and flea infestation   specific-level disorders compared to dogs that are not English Bulldogs. These included: skin fold dermatitis , tail disorder , lower respiratory tract disorder , ophthalmological disorder  and upper respiratory tract disorder . Conversely, English Bulldogs had significantly reduced odds of 3/34 (8.8%) grouped-level disorders compared to dogs that are not English Bulldogs. These were: dental disorder , spinal cord disorder  and appetite disorder   grouped-level disorders compared to dogs that are not English Bulldogs. These included: congenital disorder  has been reported for over a hundred years . This fiBoth prolapsed nictitating membrane gland and keratoconjunctivitis sicca were also reported in the current study as ultra-predispositions in English Bulldogs. A diagnosis of prolapsed nictitating membrane gland was 26.79 times more likely in English Bulldogs than in dogs that are not English Bulldogs, while keratoconjunctivitis sicca showed a less extreme but still markedly high odds ratio of 12.24. Yet, despite the greatly elevated odds ratio of prolapsed nictitating membrane gland in English Bulldogs, the 5.7% of English Bulldogs in the wider general population of dogs diagnosed with prolapsed nictitating membrane gland in this study comprise a much lower incidence than the 75% reported in previous Kennel Club surveys of pedigree English Bulldogs, as discussed above . This maThe predisposition of English Bulldogs to BOAS is well documented , 60, andMandibular prognathism constitutes a different type of ultra-predisposition in the English Bulldog population surveyed and raises the thorny question of why a conformation that would be considered a serious health issue in one dog breed can be actively selected as a desirable trait in another. As previously mentioned, ‘undershot’ jaw – the physical appearance described by the term mandibular prognathism – has been an explicit requirement of the Bulldog breed standard for over a century . AlthougGiven these common cognitive biases in owners and veterinarians towards accepting conditions as ‘normal’ in English Bulldogs that would otherwise be considered as disorders in breeds with less extreme conformations, it is therefore all the more striking that English Bulldogs are the breed with the highest proportions of predispositions among common disorders from the breeds that have been reported to date using this VetCompass methodology. English Bulldogs showed predispositions for 55.8% (24/43) of specific-level disorders. In comparison, French Bulldogs showed predispositions for 46.5% (20/43) of disorders , LabradoAlthough dogs had long been divided into loosely-recognised types, the concept of clearly differentiated and strictly delineated breeds was an invention of the Victorian era . TraditiAll breeds, by definition, are different in some ways from the average for the canine population and therefore are likely to show some breed predispositions to, and protections from, disease , 8. For Over recent years, The Kennel Club has made concerted efforts to alleviate drivers for extreme conformation from the show-ring. Since 2012, The Kennel Club has identified the English Bulldog as a breed at particularly high risk of conformation-related disease: the breed is currently grouped in Category 3 on The Kennel Club’s Breed Watch list, with show judges urged to prioritise health in their show-ring decisions . MoreoveThe current study had some limitations in addition to those previously reported for the application of primary care veterinary data for canine research , 77. ThiFuture work could include prospective cohort studies that compare disorder predisposition between English Bulldogs with moderate conformation and English Bulldogs with extreme conformation to evaluate potential welfare gains from conformational change within the breed. Repeating the current study design at defined intervals could also monitor real-world changes in the health of the English Bulldog over time following efforts by UK national groups such as the Brachycephalic Working Group .In providing further evidence that English Bulldogs experience unusually high levels of disease and show multiple predispositions and ultra-predispositions to disease, much of which is intrinsically related to their conformation, this study broadly confirms long-standing assertions that the health and welfare of English Bulldog is heavily compromised. Yet, despite extensive evidence and wide dissemination of the health issues and their serious negative welfare impacts for these dogs, it seems that many prospective owners are still not discouraged from English Bulldog ownership , 42. Eve


Sphingobacterium sp. PCS056 and Pseudarthrobacter sp. SSS035. Both isolates demonstrated the most crystal production as well as the most morphologically dissimilar crystal shapes in broth culture and were found to produce crystals as previously observed in both agar and broth media. The results from this study are consistent with the involvement of urease-positive bacteria isolated from the ICC in the formation of cave speleothems. 16S rRNA sequencing revealed a diverse set of microbes inhabiting the speleothems that have urease activity. Whole-genome sequencing of the two chosen isolates confirmed the presence of urease pathways, while revealing differences in urease pathway structure and number. This research contributes to understanding microbial-associated cave formation and degradation, with applications to cave conservation, microbiota composition, and their role in shaping the cave environment.Caves are extreme, often oligotrophic, environments that house diverse groups of microorganisms. Many of these microbes can perform microbiologically induced carbonate precipitation (MICP) to form crystalline secondary cave deposits known as speleothems. The urease family is a group of enzymes involved in MICP that catalyze the breakdown of urea, which is a source of energy, into ammonia and carbonate. Carbonate anions are effluxed to the extracellular surface of the bacterium where it then binds to environmental calcium to form calcium carbonate which then continues to grow in crystal form. Here, we studied bacterial communities from speleothems collected from the Iron Curtain Cave (ICC) in Chilliwack, B.C., Canada, to characterize these organisms and determine whether urease-positive (U+) bacteria were present in the cave and their potential impact on speleothem formation. The ICC is a carbonate cave located on the northside of Chipmunk Ridge, presenting a unique environment with high iron content sediment and limestone structures throughout. With six pools of water throughout the cave, the environment is highly humid, with temperatures ranging between 4 and 12°C depending on the time of year. Ninety-nine bacterial strains were isolated from popcorn (PCS) and soda straw (SSS) speleothems. These isolates were screened for urease enzymatic activity, with 11 candidates found to be urease-positive. After incubation, species-specific crystal morphologies were observed. Popcorn speleothem provided more bacterial diversity overall when compared to soda straw speleothem when examined under a culture-based method. Nearly twice as many U+ isolates were isolated from popcorn speleothems compared to soda straw speleothems. The U+ candidates were identified to the genus level by 16S rRNA analysis, and two isolates underwent whole-genome sequencing. Two novel species were identified as  In sulfuric acid dissolution, hydrogen sulfide (H2S) gas rises from rock fissures  and reacts with atmospheric oxygen to form sulfuric acid (H2SO4) that dissolves the carbonate rock to form caves  into H2SO4, thus increasing the dissolution of limestone, while Massilia from a New Mexico cave system could utilize aqueous carbohydrates originating from surface water, to produce acids as a metabolic byproduct, dissolving carbonate bedrock in the caves.Though chemical mechanisms of cave formation and cave architecture have been well researched, more recent studies have been focused on the biological mechanisms of cave formation. 2) degasses which can allow calcium carbonate precipitation  is a form of biomineralization and is a natural process of carbonate precipitation, occurring worldwide and mediated primarily by bacteria. MICP occurs through three primary pathways: biologically induced mineralization (BIM), biologically mediated mineralization (BMM), or biologically controlled mineralization (BCM) . Caves aMICP is a well-documented metabolic process which contributes to the formation of secondary cave deposits known as speleothems . One comWhen carbonate and calcium concentrations are at their saturation points, calcium carbonate crystals can form. The two most thermodynamically stable crystal structures common to MICP are aragonite and calcite  with vatureA, ureB, and ureC form a trimer of trimers  for 1 h. Samples were removed from the rotating incubator and left to stand for 5 min. A series of serial dilutions (log10) were performed with sequential dilutions ranging from 100 through to 10–4. For each dilution, 100 μL of the sample was pipetted and spread onto 22-mL agar plates (in triplicate) of the following media types: nutrient agar (NA), ten times diluted nutrient agar (d10NA), and Reasoner’s 2-A agar (R2A). Plates were then left to dry before incubation at 9°C until distinguishable colonies had formed.Fresh PCS and SSS samples were weighed to a wet weight of 1 g. Each speleothem was then transferred to a sterilized mortar and pestle and ground to a paste. The paste was transformed into a slurry by adding 2 mL of distilled H2H3O2)2] to one of the following: calcium nitrate (CaNO3), calcium citrate [Ca3(C6H5O7)2], and calcium chloride (CaCl2) all at 0.25% w/v. The modification of the calcium salt component of the B4 medium aimed to test calcium water solubility and the availability of aqueous calcium ions to interact with precipitated carbonate on the extracellular surface of the bacterial isolates, that is, to determine whether one calcium salt increased the rate of biocrystallization, over another. Each isolated bacterium was then inoculated onto the modified B4 agar medium in 33 mm thick agar plates, to prevent premature drying due to lengthy incubation period. A container of diH2O water was placed in the incubator to increase humidity. Modified B4 plates were inoculated, streaking one isolated colony from each of the pure culture plates . Plates were inverted and incubated at 9°C until crystals formed.B4 precipitation medium  was usedTo determine urease activity, solid agar slants were prepared and poured following the manufacturer’s instructions  was made for each U+ candidate with the introduction of urea (1 M) as a nitrogen source over a concentration-dependent manner, substituting yeast extract. U+ candidates were grown in liquid B4 precipitation medium until reached log phase (∼1.0 A600 nm) and inoculated. Incubation conditions remained the same. After 12 weeks of incubation, the broth cultures were slowly poured off, laid flat, and placed on the viewing platform of a stereomicroscope . Crystals were imaged at 40× using an iPhone 11 Pro .To determine crystal formation in an aqueous environment, 40.0 mL of MICP broth [0.25% Ca.4 aqueous solution) and 16% v/v formaldehyde (∼ 0.5 mL not touching the sample) for another 2 h. The samples were inverted after 1 h and further osmium-fumed over 48 h. Then, samples were mounted using a bulk sample holder and coated with 12 nm of gold using a Cressington 208 HR sputter coater . Samples were then imaged at various accelerating voltages, modes, and working distances on a Hitachi S4700 FESEM .Popcorn and soda straw speleothem samples were sent to the BioImaging Facility at the University of British Columbia, Vancouver, Canada for Scanning Electron Microscopy (SEM). Samples were freeze-dried and osmium-fumed in their storage tubes for approximately 1 h, and then transferred to polypropylene petri dishes with a saturated filter paper (4% w/v OSOTaq DNA Polymerase (New England Biolabs), 0.5 mM dNTPs, 0.16 mM bovine serum albumin, 0.5 μM forward primer 5′-GTG CCA GCM GCN GCG G, and 0.5 μM reverse primer 5′-GGG TTN CGN TCG TTG. Cycling conditions were 95°C for 2 min, 25 cycles of 95°C for 1 min, 68°C for 45 s, and 68°C for 45 s, with final elongation at 68°C for 5 min. The resulting PCR product was Sanger-sequenced at Génome Québec . The sequencing results were trimmed, and genus-level identification was performed using NCBI Blast . WGS was performed by Integrated Microbiome Technologies  on the PacBio Sequel II instrument. Genome analyses were performed in the Galaxy platform . Genomes2O to form a slurry of similar consistency to PCS. Speleothem pH was determined using a FieldScout SoilStik pH meter . SSS and PCS were also ground into fine powder and shipped to ALS Global Laboratories to perform inductively coupled plasma mass spectrometry (ICP-MS) total elemental analysis of sixteen elements, including biologically relevant elements such as magnesium and calcium.Both PCS and SSS were ground into a slurry to determine their relative pH. Speleothem samples were ground using a sterile mortar and pestle into a fine powder (for SSS) and a wet slurry (for PCS). To the ground, SSS was added 6 mL of diHA total of 99 isolates were grown successfully in the lab setting. A total of four SSS isolates and seven PCS isolates exhibited urease activity of varying degrees while incubating at 9°C. The 11 total urease-positive isolates, henceforth known as U+ candidates, represent 8.2% of culturable sample size. After 62 days from incubation, agar plates inoculated with U+ candidates demonstrated crystal formation on three of the four modified B4 media types. U+ candidates from both PCS and SSS grew on all media types; however, no growth was detected on B4 agar plates supplemented with calcium acetate for SSS U+ candidates only. Both PCS and SSS U+ candidates demonstrated crystal formations with varying morphologies. For the aqueous B4 experiment, broth cultures revealed crystal formations on the inside face of test tubes of three PCS and one SSS U+ candidates, respectively. For both the agar plate and aqueous B4 assays, U+ candidates, similar crystal morphologies, were seen, as demonstrated in A total of six micrographs were produced for both PCS and SSS by the Bioimaging Facility at the University of British Columbia . A totalPseudarthrobacter sp. SSS035 and Sphingobacterium sp. PCS056 underwent WGS by PacBio Sequel II. The resulting reads were assembled using Flye and annotated using PATRIC. Pseudarthrobacter sp. SSS035 was assembled into three contigs with lengths of 4,611,850, 272,440, and 149,359 nucleotides. Average coverage of each contig was over 400×, with the average coverage of the three contigs being 537×. The total genome size was determined to be 5.03 Mbp and consisted of 64.86% GC content. PubMLST determined this isolate has an 82% match to Pseudarthrobacter sulfonivorans, indicating that this is likely a previously uncharacterized species of Pseudarthrobacter. Phylogenetic analysis of the most similar organisms by genome  in addition or in replacement of nickel-dependent enzymes due to limited nickel levels in comparison with iron  U+ candidates from the ICC were found in the Australian caves, further cementing the idea that the microenvironment  may be an important selective pressure, favoring these genera. To corroborate these findings, the results from a study by While bacterial abundance may allow us to understand the community composition, we must not forget the role the pH plays in both the geological and biological worlds. We have a good understanding of how caves form, and the role that pH plays; however, we do not have a great understanding of the role pH plays in the microenvironment that cave bacteria inhabit. In this study, PCS was determined to be slightly acidic pH, while SSS was alkaline. With these findings, PCS likely undergo passive influx of calcium ions and SSS undergo active extrusion of calcium ions for calcium carbonate precipitation as demonstrated in a study conducted by in vitro experiments vs. cave environments differ greatly, discrepancies in the observed shapes of crystal made by different single isolates vs. mixed cultures were observed. Hence, we are convinced that the makeup of bacteria may influence the diversity in crystal morphology and may be related to the macroscopic shape of the speleothems themselves, suggesting that crystal budding from urease-positive candidates may influence a larger exponentiation of the crystal growth, aided along by either evaporation pattern and/or dripping of the groundwater saturated with mineral contents, to form the macroscopic speleothems that we see today.Lastly, it is imperative to understand the role that solid mineral surfaces of speleothems and their aqueous environments play in the physical geological formation of crystals. Calcium carbonate crystal formations exist as either calcite, aragonite, or vaterite. Here, we employed petrographic microscopy to discern what crystal shapes were formed from MICP of U+ candidates, and whether the crystal shapes differed between the solid and liquid MICP assays. A wide array of crystal morphologies was found upon imaging U+ candidates from both liquid and solid MICP assays, as demonstrated in Since speleothem formation is very complex and time-consuming process, lots of metabolic pathways and steps involved in cave speleothem formation remain unknown. From our lab-based crystal formations found in U+ candidate MICP assays, we now have many more questions about it than we started off. For example, we wonder whether the growth of speleothems and their structures are first nucleated at the extracellular surface of cave bacteria that precipitate calcium carbonate. How about the pH role and impact in an overall crystal formation relating to the bacterial cell membrane and subsequent cascading of proton gradients, and its effects in the microenvironment? These questions warrant us to look further into how bacterial diversity and activity influence or are influenced by the cave speleothem dynamics.Prospects include determining the nucleation role of cave bacteria in speleothem growth by performing carbonatogenic yield experiments to better understand growth kinetics of U+ candidates, and their quantitative contribution to overall speleothem formation. Moreover, determining the quantity of nucleation sites on these candidates is imperative but requires careful assessment of the number of urease enzyme copies found in their genomes and the available surface area to act as nucleation sites. Determining surface area nucleation sites requires further imaging with SEM when bacteria are in their ureolytic metabolic state. Lastly, to confirm the crystal structure being produced by U+ candidates is indeed the calcite form of calcium carbonate, X-ray diffraction microscopy must be employed.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/NC, SL, and RM obtained funding. NC, KK, and RM conceived the study and designed the experiment. KK, RM, LC, and SL conducted the experiments. KK, RM, JG, and NC designed the MS workflow. KK, RM, SL, and JG were instrumental in collecting the MS data sets. KK, RM, and JG drafted the manuscript. NV and DH assisted with microscopy. NC, NV, KK, RM, SL, and JG read and edited the manuscript. CB was JG’s Ph.D. supervisor. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Pancreatic cancer (PC) is burdened with a low 5-year survival rate and high mortality due to a severe lack of early diagnosis methods and slow progress in treatment options. To improve clinical diagnosis and enhance the treatment effects, we applied metabolomics using ultra-high-performance liquid chromatography with a high-resolution mass spectrometer (UHPLC-HRMS) to identify and validate metabolite biomarkers from paired tissue samples of PC patients. Results showed that the metabolic reprogramming of PC mainly featured enhanced amino acid metabolism and inhibited sphingolipid metabolism, which satisfied the energy and biomass requirements for tumorigenesis and progression. The altered metabolism results were confirmed by the significantly changed gene expressions in PC tissues from an online database. A metabolites biomarker panel (six metabolites) was identified for the differential diagnosis between PC tumors and normal pancreatic tissues. The panel biomarker distinguished tumors from normal pancreatic tissues in the discovery group with an area under the curve (AUC) of 1.0 . The biomarker panel cutoff was 0.776. In the validation group, an AUC of 0.9000 (95%CI = 0.782–1.000) using the same cutoff, successfully validated the biomarker signature. Moreover, this metabolites panel biomarker had a great capability to predict the overall survival (OS) of PC. Taken together, this metabolomics method identifies and validates metabolite biomarkers that can diagnose the onsite progression and prognosis of PC precisely and sensitively in a clinical setting. It may also help clinicians choose proper therapeutic interventions for different PC patients and improve the survival of PC patients. Pancreatic cancer (PC) is one of the deadliest solid malignancies, and it has an extremely low 5-year survival rate of <5% and is expected to be the second leading cause of cancer death by 2030 –3. SinceMetabolic reprogramming for tumorigenesis has been recognized as a hallmark of PC –15. AfteHere, metabolomics was applied to unveil the metabolic signature of PC from tumor tissues with paired para-carcinoma tissues and normal pancreatic tissues. We aim to assess the performance of the tissue biomarker signature to distinguish patients with PC and to predict the prognosis of PC patients.Matched pairs of pancreatic cancer  were obtained from 35 patients undergoing curative resection at the First Affiliated Hospital of Dalian Medical University. The study was approved by the Ethics Committee of the First Affiliated Hospital of Dalian Medical University with approval number PJ-KS-KY-2021-203. All samples were freshly frozen and stored at −80°C before metabolomics analysis.g for 15 min. Finally, 700 µl of the lipid extract was transferred from the upper layer to the new centrifuge tube, and 400 µl of the polar extract was transferred from the lower layer to the other centrifuge tube.Each tissue sample was taken nearly 20 mg into the grinding pipe, and the specific weight was recorded. Then, 300 µl of methanol was added, and the grinding steel beads were put into each tube. The tissue was ground for 350 s at 120 Hz. Then, 900 µl of methyl tert-butyl ether (MTBE) was added into the tube and vortexed for 5 min, followed by the addition of 250 µl purified water; then, the mixture was shaken well for 10 min at room temperature. The mixture was kept at 4°C for another 10 min to facilitate stratification and then centrifuged at 13,000 g for 10 min. Taking the 200 µl lipid layer and 200 µl polar layer as before, they were transferred into two different 2-ml centrifuge tubes (new) and used as quality control (QC) samples. Lastly, all samples were concentrated and dried by vacuum centrifugationAll the remaining extracts were mixed in each sample tube evenly and then centrifuged at 10,000 g for 15 min (4°C). Supernates of polar extracts (90 µl) and lipid extract (70 µl) were taken for detection. The detailed methods were as described before . Before analysis, polar extracts were accurately added with 100 µl acetonitrile–water complex solution , lipid was extracted with 80 µl acetonitrile–isopropanol solution , the mixture was vortexed for 5 min, and it was centrifuged at 13,000 d before .2 hold change >1.3 or <−1.3.Pathway enrichment was conducted on differential metabolites between pancreatic tumor tissues and paired normal pancreatic tissues. We also searched The Cancer Genome Atlas (TCGA) and the Genotype-Tissue Expression (GTEx) database and analyzed the expressions of key genes in the enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways from PC patients. Combined with the results that we achieved from the online database, we selected pathways that have both differential genes and metabolites between pancreatic tumor tissues and paired normal pancreatic tissues. Then, we chose metabolites that belong to these pathways and had logTissue samples were randomly divided into two groups: establishment group and validation group. There were 20 normal pancreatic tissues and 20 pancreatic cancer tissues in the establishment group, while there were 11 normal pancreatic tissues and 15 PC tissues in the validation group. The least absolute shrinkage and selection operator (LASSO) algorithm analysis was used to confirm the independent predictors and build a metabolite panel for differentiating pancreatic tumor tissues and normal pancreatic tissues. The area under the curve (AUC) was used to estimate the performance of the discriminant model. The Youden index (J=sensitivity+specificity−1) was calculated in conjunction with binary logistic regression. We tested the ability of the discriminant model to predict the prognosis of PC patients by log-rank test and Cox regression. Univariate analysis was statistically significant at a p-value <0.10 and was entered into a multivariable Cox proportional-hazards model.http://www.metaboanalyst.ca/MetaboAnalyst/). Transcriptomic data of PAAD samples were downloaded from the TCGA data portal  and GTEx . Gene set enrichment analysis has been performed on MSigDB Collections (https://www.gseamsigdb.org/gsea/msigdb). Unpaired t-test, binary logistic regression, log-rank test, and Cox regression analyses with OS were performed using SPSS 26.0 software . Functional enrichment metabolic pathway analysis of metabolites was performed using MetaboAnalyst. The LASSO algorithm, heatmap, bar, and nomogram plots were conducted on R studio (version 3.6). Kaplan–Meier curves were conducted on GraphPad Prism 8.0. All of the p-values involved in this study were two-tailed probabilities. The difference was statistically significant for p <0.05.The metabolites that were consistently detected in at least 80% of the samples were included in the statistical analysis. Multivariate statistical analyses orthogonal partial least squares discrimination (OPLS-DA) and network analysis were performed using the open-access software Metaboanalyst 4.0  that were observed to metabolic differentiate between the PC tumors tissue and the normal tissues  and paired normal pancreatic tissues (n = 20) collected from the PC patients were comparatively analyzed. Substantial metabolic changes were observed using a supervised OPLS-DA model to distinguish the tumor tissue from the normal controls sensitively. This model achieved 0.783 for Q2 (p<0.001) and 0.901 for R2Y (p<0.001) with 1,000 permutation tests . The details of these significantly different metabolites are listed in Finding metabolite panel biomarkers for precise diagnosis has a decisive impact on patient treatment and survival. According to the pathway enrichment results, we first randomly separated the tumor and normal tissue samples into two groups, namely, the model establishment group and validation group  for PC patients. First, logistic regression values g(z) for all the 35 patients, both in the discovery and validation set, were obtained according to the six-metabolite discriminant model   Table 2.In summary, we have uncovered the metabolic pathway alterations and the changing pattern among pancreatic tumors tissue, para-carcinoma tissue, and normal pancreatic tissues. We identified a metabolite panel of biomarkers [kynurenic acid, hippuric acid, gamma-aminobutyric acid, PC(36:2)-PC(18:0/18:2), uridine 2′,3′-cyclic phosphate, and 5′-methylthioadenosine) that can precisely diagnose pancreatic cancer by efficiently distinguishing between pancreatic tumors tissues and normal pancreatic tissues. Importantly, the metabolite panel was significantly associated with the overall survival of PC patients and can serve as a good prognostic marker for PC.Earlier studies demonstrated that reprogrammed glucose, amino acid, and lipid metabolism in the tumor microenvironment and metabolic crosstalk contribute to the unlimited progression of pancreatic tumors –23. EnhaThe widely rewired amino acids metabolism was also observed in pancreatic tumor tissue and para-carcinoma tissue when compared with normal pancreatic tissue. Emerging evidence has revealed that amino acid metabolism plays an important role in PC initiation and progression –33. ConsCombining the metabolites and gene pathway enrichment results, we selected six metabolites as a panel to be a discriminant model. The panel contained metabolites from different chemical classes including lipid, amino acid, pyrimidine, and microbial metabolites. The metabolites panel achieved an ideal differentiating effect between the tumor and normal pancreatic tissue. This is suggestive of the reliability and representativeness of the select metabolites in the panel, which could be what underlies the tumorigenesis of PC. Normally, PC patients who undergo radical surgical resection had a comparable better OS, with 5-year OS rate reaching 20%. In our study, PC patients have f(x)<0.776 ; the mOS was 28.0 months and intriguingly comparable with that of radical surgical resection. These results demonstrated that our discriminant model had clinical predictive value for PC patients. However, this measurement based on tissue samples is not applicable for those who did not receive surgical resection or are on drug therapy. Therefore, it is still necessary to integrate widely accepted clinical parameters and biomarkers for a comprehensive prognostic risk assessment.In this study, metabolic classification based on the metabolic profiles of PC tissues provided novel insights for the heterogeneity of PC and could be performed as a new method to explore the prognosis. Additionally, the association of the metabolites panel with OS of pancreatic cancer patients indicated its potential in guiding medication and treatment, which may have the capacity to monitor PC from a metabolic view and guide clinicians in PC prevention and treatment.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by the Ethics Committee of the First Affiliated Hospital of Dalian Medical University. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.JL, SD, and PY conceived and designed the experiment. CL, HQ, MS, and HHL performed the experiments. HYL, TW, ZW, and AW analyzed the data CL. HQ drafted the manuscript. JL, SD, and PY reviewed the manuscript. CL and PY edited the final manuscript. All authors contributed to the article and approved the submitted version.This work was supported by the key research and development project of Liaoning Province (No. 2018225054), National Key Research and Development Program of China (No. 2018YFE0195200), the National Natural Science Foundation of China (No. 81873156), and Young Science and Technology Talent Project of the Education Department of Liaoning Province China (No. LZ2020075).We thank the Key Laboratory of Integrative Medicine, the First Affiliated Hospital of Dalian Medical University, for experiment support and the iPhenome Biotechnology (Yun Pu Kang) Inc., Dalian for data generation.ZW and PY are co-founders of iPhenome (Yun Pu Kang) Biotechnology Inc. ZW is an employee of iPhenome (Yun Pu Kang) Biotechnology Inc.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.