Toughness of epoxies is commonly improved by adding thermoplasticphases, which is achieved through dissolution and phase separationat the microscale. However, little is known about the synergisticeffects of toughening phases on multiple scales. Therefore, here,we study the toughening of epoxies with layered poly(ether imide)(PEI) structures at the meso- to macroscale combined with gradientmorphologies at the microscale originating from reaction-induced phaseseparation. Characteristic features of the gradient morphology werecontrolled by the curing temperature (120–200 °C), whilethe layered macro structure originates from facile scaffold manufacturingtechniques with varying poly(ether imide) layer thicknesses (50–120μm). The fracture toughness of the modified epoxy system isinvestigated as a function of varying cure temperature (120–200°C) and PEI film thickness (50–120 μm). Interestingly,the result shows that the fracture toughness of modified epoxy wasmainly controlled by the macroscopic feature, being the final PEIlayer thickness, i.e., film thickness remaining after partial dissolutionand curing. Remarkably, as the PEI layer thickness exceeds the plasticzone around the crack tip, around 62 μm, the fracture toughnessof the dual scale morphology exceeds the property of bulk PEI in additionto a 3 times increase in the property of pure epoxy. On the otherhand, when the final PEI thickness was smaller than 62 μm, thefracture toughness of the modified epoxy was lower than pure PEI butstill higher than pure epoxy (1.5–2 times) and “bulktoughened” system with the same volume percentage, which indicatesthe governing mechanism relating to microscale interphase morphology.Interestingly, decreasing the gradient microscale interphase morphologycan be used to trigger an alternative failure mode with a higher cracktortuosity. By combining facile scaffold assemblies with reaction-inducedphase separation, dual-scale morphologies can be tailored over a widerange, leading to intricate control of fracture mechanisms with ahybrid material exceeding the toughness of the tougher phase. Unfortunately, for some tougheners, the additionof the second phase into the epoxy system also reduces overall modulusand limits the end-use temperature of the system.5 Moreover,the viscosity of the resin system may also increase significantlyby bulk resin modification, primarily through thermoplastics, whichmakes them unsuitable for liquid composite molding processes.6Epoxy-based compositesare subjected to high static and dynamicloadings in engineering applications, which require higher resistanceto fracture. In contrast, epoxies with high cross-linking densitiesare inherently brittle and typically have a low fracture toughness.However, different approaches are known to increase their fracturetoughness.8 Combining theseeffects results in hierarchically toughened structures. The firstand most relevant approach for toughening on a macro scale is theintroduction of ductile interlayers into a brittle material, whichleads to toughening through the inhomogeneity effect.10 The material inhomogeneity effect is based on spatial variationsin material properties significantly influencing the crack drivingforce, i.e., a material inhomogeneity can hinder or promote crackpropagation. The ductile interlayers act as crack arresters, so thatthe fracture toughness of the base material strongly increases. Ifthe ductile interlayers are thin enough, the loss in strength is almostnegligible.11 For instance, Sistaniniaet al. focused on designing new fracture resistance material by utilizationof the material based on the yield stress inhomogeneity effect.12 They used ductile interlayers (low-strengthsteel) to toughen the high-strength steel matrix and reported a criterionof optimum architectural parameters, i.e., optimum interlayer spacing.The multilayer specimen’s fracture toughness was at a similarvalue to the sample having a single interlayer in the case of interlayerspacing larger than the process zone around the crack tip. Likewise,Zechner and Kolednik used aluminum sheets as multilayers with a polymeradhesive.13 Structures in different configurations are tested. The resultsshowed that the crack arrester configuration exhibits a tremendouslyimproved crack growth resistance (73.7 kJ/m2) and fractureinitiation toughness compared to the single sheet configuration (33.6kJ/m2), which was attributed to the so-called “materialinhomogeneity effect”.13 Severalother studies introducing soft interlayers into brittle material showedincreased fracture toughness due to the inhomogeneity effect.15 All of these studies highlighted the importance of material inhomogeneityat specific scales in improving the fracture toughness of brittlematerial. This concept is also adopted in this current study to toughenan epoxy system  by using multilayers of poly(etherimide) , leading to the material inhomogeneityeffect .In contrast to bulk modification, severalother approaches andeffects to toughen a brittle material have been observed on the macro-,the micro-, and/or the nanoscale.19 For instance, Harismendy et al.,reported the formation of a particulate morphology by adding 10 wt% PEI into the epoxy which resulted in a 2-fold increase in fracturetoughness due to crack path deflection mechanism.16 Likewise, as reported in our previous study,20 in the case of using a thermoplastic film asthe ductile interlayer, liquid reactive thermoset monomers diffuseinto the glassy thermoplastic and partially swell or dissolve it,which results in the diffusion of TP polymeric chains into the epoxyresin. The mutual diffusion of the components creates a concentrationgradient in the interfacial region.21 Theproceeding cure reaction between the comonomers induces a phase separationleading to a gradient morphology in the interfacial zone.22 The gradient interphase shows different morphologiesthroughout its thickness depending on the concentration of epoxy andPEI. This granular morphology can impart toughening effects similarto those of micro- and nanosized dispersed particles, as found inbulk toughening of thermosetting systems. These “inclusions”can induce various energy dissipation mechanisms, thereby limitingthe onset and propagation of cracks by different toughening mechanisms,i.e., crack deflection, crack pinning, or debonding of particles.24 The cure profile strongly affects the thickness of the interphaseand its morphology.20Another potentialtoughening effect occurs at one scale lower,at the microscale. Several researchers have reported the microtougheningof epoxy using a thermoplastic (PEI).The aim ofthis study is to understand the effect of micro- andmacrotoughening approaches on the fracture toughness of an epoxy systemby using thermoplastic PEI multilayers. Thereby, the following researchquestions are addressed: (i) what are the respective effects of macrotoughening and microtoughening  on the fracture toughness of epoxy? (ii) Is there any synergeticeffect between these two toughening mechanisms? From the morphologicalpoint of view, it is not known whether a larger scale heterogeneityin the submillimeter range, by having thermoplastic multilayers, wouldpossibly be more effective. Therefore, the first research questionis addressed by toughening the epoxy resin using multilayers of PEIwith varying thicknesses (50–120 μm) to tune the macrotougheningphenomenon. The second research question is investigated by developingdiffusion-controlled interphase gradients (micron-sized morphology)between the aforementioned multilayers at the epoxy/PEI interfaceas a function of different cure temperatures (120–200 °C)and determining the resultant fracture toughness of the system. We,therefore, present a method to create dual-scale morphologies by (a)assembling multilayer scaffolds of thermoplastic tougheners with architectedporosity (giving rise to the morphology in the submillimeter range)and (b) creating diffusion-controlled interphases (micron-sized morphology)along with the reaction-induced phase separation after infiltrationwith an epoxy system. This allowed us to discriminate the effect ofthe thickness of the thermoplastic layer, cure temperature, interphasethickness, and morphological features on the fracture toughness ofthe resultant hybrid material.22.1n ≈90, Tg = 217 °C), with differentthicknesses , provided by SABIC, SaudiArabia, were used as the tough phase. The thermoset resin was preparedas a blend of M--N,N-bisaniline , and bisphenol-Fepoxy resin monomer . 3,3′-sulfonyldianiline was used as a curing agent for epoxy. Huntsman,Switzerland, supplied all these chemicals.Thin films of PEI  was then incorporatedinto 50 g of epoxy resin. The prepared mixture was stirred with aspeedmixer at 1200 rpm for 4 min. The epoxy resin was degassed byusing vacuum for 5 min to remove the entrapped air bubbles.2.2.2PEI scaffoldswere prepared by stacking the PEI films periodically in a 1:2 ratioof equal thickness as “PEI layer”, “spacer”,“spacer”, “PEI layer”, “spacer”,“spacer”, “PEI layer”, “spacer”,“spacer”, and so on 1 to obta2.2.325 The timeand temperature during the curing stage  cure temperatures of 120,140, 160, 180, and 200 °C were selected to attain at least 70%degree of cure at the first dwell. The postcure time and temperaturewere selected to attain full cure of the sample.In this study, the PEI-toughenedepoxy samples were cured using a two-dwell cure cycle approach alreadyreported in the literature.2.2.426 The dimensions of the SENB sample were as follows:length = 26.4 mm, width = 6 mm, and thickness = 3 mm.The fracturetoughness of the specimens was determined by single-edge notched bending(SENB) testing according to the ASTM D5045-99 standard.a0) with the help of a saw blade of 0.12 mm thickness. Then, the notchwas filled with diamond paste ; a razor blade wasinserted in the notch, and a natural crack (Δa) was obtained by sliding the razor blade (see As shownin lade see 2.a0 + Δa) was ±1.7mm. All ofthe tests were conducted on a Zwick/Roell tensile test machine witha 1 kN load cell and a 10 mm/min testing speed. The device was setup according to the ASTM standard D5045-99.26 The resulting load–displacement curves were then translatedto KIc and GIc values. The plastic zone around the crack tip in our system wasestimated to be 62 μm by the Irwin model27 under the plane strain condition (see the Supporting Information). Therefore, linear elastic fracturemechanics (LEFM) was applied to the system because the radius of theplastic zone was smaller compared to the crack and ligament length.27The total length of the notch and natural crack was used to etch the polishedsamples following the procedure explained elsewhere.20 The etched samples were coated with gold using a sputtercoater and then analyzed using SEM. For fractography analysis, thecross-section of fractured samples obtained after SENB testing wasalso coated with gold and analyzed using SEM.The interphaseand morphology of the cured PEI toughened epoxy samples were studiedby using field emission scanning electron microscopy (SEM)  and confocal laser scanning microscopy (CLSM) . For morphological analysis, samples were embedded in afast-cure epoxy resin, followed by grinding and polishing. 33.120 The size of these epoxy droplets was observed to decrease graduallytoward the pure PEI region due to the increase in PEI content. Similarresults were also obtained for the samples cured at 140, 180, and200 °C.The interphaseformation between epoxy and PEI at different cure temperatures wasanalyzed by using CLSM and SEM. An example of the multilayer sampleconsisting of a PEI layer of 90 μm cured at 160 °C (1stdwell temperature) is shown in Most of the samples showed a distinct gradientmorphology. However,two cure conditions showed different behaviors: (1) samples curedat 120 °C and (2) samples cured at 200 °C with a low PEIlayer thickness. For the first case, no visible gradient morphologywas observed for samples cured at 120 °C at the given resolutionfor all of the studied PEI layer thicknesses. For example, the SEMimage of a sample with 60 μm PEI film cured at 120 °C isshown in On the other hand, the maximum interphase thicknessfor a 50 μmPEI layer seems to be found at 200 °C, which, however, was linkedto the complete dissolution of the monolithic PEI layer and difficultyin analyzing the thickness of a single interphase, which would behalf of the measured value. Hence, in the case of the 50 μmPEI layer, the maximum interphase thickness was evident at a curetemperature of 180 °C. This behavior shows that the criticalcure temperature required for achieving maximum interphase thicknessshifted toward a higher value as a function of decreasing PEI thickness.Furthermore, this graph indicates that the higher the PEI layer thickness,the higher the interphase thickness at any cure temperature. For instance,the interphase thickness is 72 μm for sample 12P14 with a PEIlayer thickness of 120 μm, while for the same cure conditions,with a PEI layer thickness of 50 μm (sample 5P14), it is 33.4μm.3.220 are plotted asa function of position along the interphase for samples cured at differenttemperatures .The morphologygradient consists of epoxy-rich droplets in a continuous PEI-richphase. The epoxy droplet size and frequency, calculated from blobdetection with OpenCV,eratures 6. At 120eratures 6a, a nareratures 6b, indic3.3For SENB of multilayersystems, the precrack tip location in the tough PEI or brittle EPphase influences fracture behavior significantly. In S3. It is witnessed that in the case of 6P12,crack initiation happened in the epoxy phase (step 1A) as the cracktip was in the epoxy phase, which caused an increase in the load untilstep 1B. After that, a sharp decrease in load is found as the crackpasses through interface 1 . The solidline in KIc value calculated accordingto the rule of mixtures16epoxy = 2/3 and ϕPEI = 1/3 represent the volume fractions of epoxy and PEI,respectively. The rule of mixture represents the bulk resin modification,and therefore, The effect of cure temperature and PEI film thicknesson the fracturetoughness and critical energy release rate of samples is shown in KIc) and energyrelease rate (GIc) displayed a decreasingtrend with the increase in cure temperature. The increase in the planestrain fracture toughness, KIc, and energyrelease rate, GIc, compared to pure PEI,particularly at the lower cure temperatures (120–140 °C),strongly suggests the synergistic benefit of multilayer architectureand morphologies present at the interphase.Both fracture toughness . At 140 °C, the remainingPEI layer thickness was similar to the plastic zone size in the caseof the 90 and 120 μm initial PEI layer, while the remainingPEI layer thickness for the 50 and 60 μm initial PEI layer wasless than the plastic zone. At higher cure temperatures, the remainingPEI layer thickness was well below the plastic zone size for all ofthe initial PEI thicknesses.The value of 4The enhancement in fracturetoughness of epoxy toughened with PEImultilayers is observed on two levels, i.e., (1) macroscale and (2)microscale.4.128 found an increase in strain energy when a crack grows from a brittleto a ductile material, called the antishielding effect. On the otherhand, the strain energy is diminished when a crack grows from theductile to the brittle phase. This so-called shielding effect canstop the crack from growing entirely, leading to crack arrest at theinterface.The macro-scale tougheningof epoxy comes from introducing tough and ductile PEI layers intothe epoxy, which creates the inhomogeneity effect . Several studiestcritical) is equal to the plastic zone size.29 At a low cure temperature . Therefore,this result shows that the remaining PEI thickness  plays a dominant rolein defining the macrotoughening phenomenon, where the critical valuewas found to be in the range of the plastic zone of the PEI, about∼60 μm.It is reported that the maximum inhomogeneity effectis achieved when the thickness of the interlayer . There are three regions of interestin this morphology: (1) PEI inclusions in the epoxy-rich phase, (2)the interphase region (with or without the gradient morphology dependingupon the cure temperature), and (3) the PEI-rich phase with epoxyinclusions. The epoxy inclusions in the PEI-rich phase were not discussedin the following sections due to their negligible effect on the fracturebehavior of samples. Therefore, the microscale toughening of epoxywas explained based on two remaining regions: (i) gradient morphologyin the interphase region and (ii) PEI inclusions in pure epoxy.As already discussed,liquid reactive thermoset monomers diffuse into the glassy thermoplasticand partially dissolve it. This mutual diffusion of the componentscreates a gradient morphology in the interfacial region and cured at 200 °C showed a complete dissolution of the PEIlayer  at a cure temperature of 120 °C 6a, suggeeratures6b, whichEIlayer 4b.In thrulesee 10a,whicThe size of the epoxy particles in the gradientinterphase governstheir connectivity with the thermoplastic phase. 32 A crack generally propagates in the direction of the least resistance.Therefore, at lower temperatures, the crack prefers to propagate aroundthe interface instead of propagating through the interface due tothe weak adhesion , the interphase typically shows the presenceof only bigger particles  calculated by Griffith’stheory.cles see 4a, which4.2.234 The fracture surfaces of the epoxy-rich phase with PEI inclusionsshow different toughening mechanisms at different cure temperatures.The epoxy-rich phase shows a ductile deformation (3D pattern) in thecase of low cure temperature , strongly suggests the dominanceof the macrotoughening phenomenon coming from multilayer architecture(To identify the main controlling parameter (cure temperature orPEI film thickness) for modifying the fracture toughness of epoxy,plane strain fracture toughness, ring see 14. This itecture14. This36 Furthermore, by decreasing the final PEI film thickness, the mechanicalproperties of PEI-toughened epoxy approach the theoretical valuesof KIc and GIc .On the other hand, when the final PEI thicknessis smaller than62 μm, the fracture toughness of modified epoxy is lower thanpure PEI but still higher than pure epoxy and “bulk toughened”systems with the same volume percentage, which shows that the governingmechanism, in this case, is microtoughening. This mechanism mainlycomes from the presence of bigger epoxy particles in gradient morphology,which plays a significant role during fracture  and gives higher crack resistance. Moreover, with a decreasein the final PEI thickness, the spacing between the two PEI layersincreases, eventually resulting in lower fracture toughness. Likewise,Kolednik et al. and several other studies found that the lower thespacing between two interlayers, the more the fracture toughness forthe given thickness. and GIc 14 obtain and GIc 1. In litKIc) as a function of processing parameters (cure temperature), morphology(interphase thickness), and architectural configuration (thicknessof the PEI film). This graph shows that a lower interphase thicknessresults in better fracture toughness of the epoxy system and PEI film thickness (50–120 μm). PEI scaffolds wereprepared by stacking the PEI films periodically in a 1:2 ratio ofequal thickness, which resulted in scaffolds with 33 vol % of PEIand 66 vol % of epoxy. This study cured the PEI-toughened epoxy samplesusing a two-dwell cure cycle.1/2) at higher final PEI film thickness (95 μm).However, changing the initial PEI film thickness or cure temperaturecan control this final PEI film thickness. Compared to pure PEI, thehigher fracture toughness of toughened epoxy, particularly at thehigher final PEI film thickness (larger than 62 μm), stronglysuggested the occurrence of both macro- and microtoughening. However,the dominant mechanism is macrotoughening coming from the multilayerarchitecture. This dominance mainly comes from the effective inhomogeneityeffect due to the critical final PEI layer thickness. On the otherhand, the fracture toughness of modified epoxy was lower than purePEI but still higher than pure epoxy and bulk-modified with a similarvolume fraction when the final PEI thickness was smaller than 62 μm,which showed that the only governing mechanism was microtoughening.This mechanism mainly comes from bigger epoxy particles in gradientmorphology, which plays a significant role during fracture  and gives higher crack resistance. This resultsshowthat for high-temperature processing, the initial PEI film thicknesshas to be selected such that the remaining film thickness allows usto obtain the synergy between micro- and macrotoughening.The result shows that the fracturetoughness was mainly controlledby the final PEI thickness, i.e., higher fracture toughness (4.53MPa mKIc and GIc obtained from the mixture rule.This study provides an understanding of the hierarchical tougheningof epoxy using a multilayer system which can further be explored inthe field of fiber-reinforced composites for aerospace applications.Moreover,with the decrease in the final PEI thickness, the spacingbetween the two PEI layers increases, eventually resulting in lowerfracture toughness. However, in the current study, the influence ofinterlayer spacing cannot be investigated because of the fixed EP/PEIvolume ratio. Therefore, in the future, to effectively understandthe influence of interlayer spacing on the fracture toughness of epoxy,further research needs to be performed without fixing the volumetricratio between epoxy and PEI. Furthermore, by decreasing the finalPEI film thickness, the mechanical properties of PEI-toughened epoxyapproach the theoretical values of


This study was conducted to evaluate the effects of optimal trace mineral levels on the physiological responses, reproductive performance, litter performance, blood profiles and milk composition in gestating sows.Nutrient Requirements of Swine. During lactation period, all the sows were fed the same commercial lactation diet.A total of 59 multiparous sows (Yorkshire×Landrace) with similar body weight (BW), backfat thickness (BF), and parity were assigned to one of four treatments with 14 or 15 sows per treatment using a completely randomized design. The treatments were 100% (M1), 300% (M3), 600% (M6), and 900% (M9) of the National Research Council (NRC) No significant differences were observed in the BW, BF, reproductive performance, milk composition, or growth performance of the piglets. On day 70 of gestation, the serum zinc concentration showed a quadratic response to M6 treatment . Moreover, as the dietary mineral levels increased, the zinc concentration increased linearly at 110 days of gestation . Furthermore, copper and iron concentrations in the serum of sows at 24 h postpartum decreased linearly when high levels of dietary minerals were provided . In the serum of piglets, serum zinc concentrations decreased linearly , and iron concentration showed a quadratic response  with an increase in trace mineral premix levels in gestation diets.The current trace mineral requirements of NRC (2012) are suitable for gestating sows, and the addition of dietary mineral levels in the gestating diet did not show any improvements during the gestation and lactation periods. Pigs require macro- and micro-minerals for maintenance, growth, and reproduction . MacromiGenerally, the availability of trace mineral are limited and supplemented high dose are required to fulfil animal needs which often results in toxicity issues and deficiency of other trace mineral elements . As tracDietary trace mineral interactions were importantly considered to pig diet formulation, especially copper, zinc, and iron contents. In older studies, high concentrations of dietary zinc induce the intestinal metallothionein to bind copper and increase copper requirement . MoreoveTherefore, the current study was conducted to investigate the effects of different levels of dietary trace mineral premix on the physiological responses, reproductive performance, serum mineral content of sows and piglets, and milk composition during the entire gestation period.All experimental procedures involving animals were conducted following the Animal Experimental Guidelines provided by the Seoul National University Institutional Animal Care and Use Committee .A total of 64 F1 multiparous sows (Yorkshire×Landrace) with average BW of 231.5 kg, average backfat thickness of 23.3 mm, and an average parity of 4.98 was allotted to one of four treatments considering BW, backfat thickness, and parity in completely randomized design after mating. All sows took two times of artificial insemination service according to estrus cycle after weaning and checked pregnancy at day 35 of gestation by ultrasound scanner . After pregnancy diagnosis, only 59 F1 multiparous sows were pregnant and continued to consume their treatment diets. During the experimental period, multiparous sows of third or over third parity were fed a 2.4 kg/d gestation diet.The experiment was allotted to one of 4 treatments considering BW, backfat thickness, and parity in completely randomized design. All experimental diets for gestating sows were formulated based on corn-soybean meal and trace mineral premix was supplemented by treatment levels. The treatments were 100% (M1), 300% (M3), 600% (M6), and 900% (M9) of the NRC  requiremad libitum until weaning.All experimental sows (parity: 3 to 6) were fed an experimental diet once a day at 08:00 h and provided 2.4 kg/d during gestation and the gestation diet was decreased gradually 0.2 kg/d during 5 days before farrowing. After farrowing, sows were fed lactation diet of 1, 2, 3, 4, and 5 kg/d as lactating age increased and fed a diet All sows were accommodated in individual gestation stalls (2.20×0.64 m) where the indoor temperature was regulated to an average of 20°C by an automatic ventilation system. At day 110 of gestation, sows were moved from the gestation barn to farrowing crates (2.50×1.80 m) after washing and disinfecting their body, especially breast and vulva. None of the sows were treated with delivery inducer and they were assisted when dystocia occurred. The room temperature of the lactating barn was kept at 28°C±2°C and the baby house under heating lamp was kept at 32°C±2°C. The air condition of the lactating barn was regulated automatically by the ventilation system and air-conditioner. After weaning, the sows were moved to a breeding barn for the next oestrus cycle.After farrowing, piglets were cross-fostered within treatment until 24 h postpartum to balance the suckling intensity of sows with equalization of litter size, and thus to minimize any effect of initial litter size potentially affecting litter growth. Cutting of the umbilical cord and tail and castration were conducted 3 days after birth, and piglets were injected with 150 ppm Fe-dextran  injection. All piglets were not fed creep feed during the whole lactation period. Weaning was performed at approximately 24±3 d.2 position  by an ultrasound device . Daily feed wastage was recorded during lactation and lactation feed intake was measured when measuring the BW and backfat thickness of lactating sows at day 21 of lactation. The weaning to estrus interval (WEI) of sows, as an important parameter for evaluating reproductive performance, was measured after weaning.The BW and backfat thickness of sows were measured at mating, day 35, 70, and 110 of gestation, 24 h postpartum, and day 21 of lactation, respectively. The BW of the sow was measured by electric scale  for sow and backfat thickness was measured at the PAfter farrowing, the number of piglets for total born, stillbirth, mummy, alive piglets was recorded, and the BW of alive piglets, stillborn, and mummy was measured by electric scale . When measuring the BW of piglets, ear notching was practiced for the experiment.After ear notching, cross-fostering of the piglets within the same treatment was performed until 12 h postpartum for equalizing litter size. The number and BW of piglets was measured again at day 21 of lactation to calculate litter weight, piglet weight and both weight gain.2E; Becton Dickinson, UK) and centrifuged at 3,000 rpm and 4°C for 15 min  after clotting at room temperature for 30 min. The upper liquid (serum) of the blood was separated to a microtube  and stored at −20°C freezer until later analysis.Blood collection from sows based on similar BW and backfat thickness (n = 6 for each treatment) was taken by venipuncture of the jugular vein using 10 mL disposable syringes at mating, day 35, 70, 110 of gestation, 24 h postpartum and day 21 of lactation. Blood from suckling piglets (n = 12 for each treatment) was collected from the anterior vena cava using 3 mL disposable syringes at 24 h postpartum and 5 mL disposable syringes at day 21 of lactation. All blood samples were enclosed in serum tubes  as well as ethylenediaminetetraacetic acid tube  was into the blood vessels of the sow’s ear. Colostrum and milk were collected in 50 mL conical tubes  from the first and second teats. After collection, samples were stored in a freezer (−20°C) until further analysis. Proximate analysis of colostrum and milk was conducted using Milkoscan FT120 .Serum and diets were analyzed for trace mineral using the spectrofluorometric method outlined by AOAC . The anaAll collected data were carried out by least squares mean comparisons and were evaluated with the general linear model procedure of SAS . OrthogoThe effects of dietary trace mineral levels on gestating lactating sows are shown in The effects of dietary trace mineral levels on the reproductive and litter performance of the sows are shown in The effects of dietary trace mineral levels on the chemical composition of colostrum and milk in sows are shown in The effects of dietary trace mineral premix levels on serum zinc, copper, and iron concentrations in gestating sows are shown in The effects of dietary trace mineral premix levels on the concentrations of serum zinc, copper, and iron in piglets are shown in Several studies have evaluated the effects of single trace mineral levels on sow performance. Peter and Mahan  has repoNormal feed intake during lactation is closely associated with sow BW loss, milk production, and WEI ,22. SeveIn the current study, feeding with different trace mineral levels did not affect the reproductive or litter performance of sows. Some studies have shown no significant differences in reproductive performance and litter growth when a single mineral was added during late gestation ,24 and lThe nutrients in sow milk are especially necessary for suckling piglets, who can only obtain nutrients from milk , and milLimited information has been gathered regarding the complicated changes in serum mineral levels  when gestating sows are fed different dietary mineral premix levels. Kalinowski and Chavez  reportedPoulsen  reportedThe current NRC  trace mi


Despite the availability of three network meta-analyses (NMA) examining the efficacy, treatment completion, and adverse events associated with all latent tuberculosis infection (LTBI) treatments, there is currently no evidence to support the notion that the benefits of these treatments outweigh the potential risks. This NMA aimed to conduct a comprehensive comparison and update of the efficacy, treatment completion rates and adverse events associated with recommended treatment options for LTBI for individuals with confirmed LTBI, as outlined in the 2020 World Health Organization (WHO) Consolidated Guidelines for TB preventive treatment. A comprehensive search of the MEDLINE and Scopus databases was conducted until April 2023. The NMA was applied to estimate the risk difference and corresponding 95% confidence interval (CI) using a combination of direct and indirect evidence. The risk–benefit assessment was employed to evaluate the feasibility of the extra benefits in relation to the extra risks. The primary outcomes of interest in this study were active TB disease, completion rates, and adverse events. The meta-analysis incorporated data from 15 studies, which collectively demonstrated that the administration of a placebo resulted in a significant increase in the risk of developing TB disease by 1.279%, compared to the daily intake of isoniazid for 6 months (6H). Furthermore, treatment completion rates were significantly higher when using isoniazid plus rifapentine weekly for 3 months (3HP) and rifampicin daily for 4 months (4R), as compared to 6H. Considering adverse events, the combination of 3HP, 4R, and isoniazid administered daily for 9 months (referred to as 9H) significantly decreased adverse events by 4.53% in comparison to 6H. The risk–benefit assessment showed that alternative treatment regimens  had a lower incidence of adverse events, while demonstrating a higher efficacy in preventing TB, as compared to 6H. This review indicates that there were no significant differences observed among various active treatment options in terms of their efficacy in preventing active TB. Moreover, completion rates were higher in 3HP and 4R, and a reduction in adverse events was observed in 3HP, 4R, and 9H. The etiology of TB is caused by Mycobacterium tuberculosis. However, a significant proportion of individuals, approximately 90% exhibit no clinical manifestations of the disease, indicating the presence of latent tuberculosis infection (LTBI)2. It has been reported that populations with LTBI have an estimated lifetime probability of developing active TB disease of approximately 5% to 10%3. As per a meta-analysis conducted in 2019, the global prevalence of LTBI was found to be 24%4. The South-East Asia region is widely recognized for having the highest prevalence of LTBI, accounting for 35% of the global burden5.Tuberculosis (TB) is a common cause of mortality arising from a solitary infection. In the year 2019, it is estimated that there were approximately 1.2 million deaths worldwide attributed to TB disease6. According to the 2020 World Health Organization (WHO) Consolidated Guidelines for TB preventive treatment7, it is recommended to utilize either a tuberculin skin test (TST) or an interferon-gamma release assay (IGRA) for the purpose of testing for LTBI. Furthermore, the treatment options recommended for the management of LTBI irrespective of the individual's HIV status consist of the following regimens: isoniazid for 6 months daily (6H), isoniazid for nine months daily (9H), isoniazid plus rifampicin for three months daily (3HR), and isoniazid plus rifapentine for three months weekly (3HP). Furthermore, there are alternative treatment regimens available, such as a four-month daily regimen of rifampicin (4R) or a four-week daily regimen of isoniazid plus rifapentine (1HP).Despite the significant burden of LTBI in Asia, the current approach to treating LTBI is suboptimal, primarily due to low adherence rates and the occurrence of hepatotoxicity as a serious adverse event10, completion rates10, and occurrence of adverse events associated with treatment regimens for LTBI10. Two published NMAs incorporated all individuals receiving LTBI treatment10. These studies encompassed cases with or without confirmation of LTBI testing. Additionally, another study specifically examined populations with confirmed LTBI, focusing solely on the efficacy and completion rate of LTBI regimens, without investigating adverse events9. Moreover, it is worth noting that the treatment regimens mentioned in all the published NMAs encompassed several regimens, including isoniazid for durations ranging from 9 to 72 months and regimens containing pyrazinamide (PZA) for the purpose of indirect comparison. It is important to highlight that these regimens did not currently align with WHO recommended LTBI treatment due to their toxicity. Furthermore, based on the 2020 WHO Consolidated Guidelines7, it is imperative to obtain robust evidence to ascertain that the benefits of recommended TB preventive treatment outweigh any potential risks to individuals with LTBI. However, until recently, such evidence has not yet been available. Accordingly, the objective of this NMA was to evaluate whether the extra efficacy (benefit) of LTBI treatment is worth the extra adverse events (risk) using the risk–benefit assessment and to provide an updated analysis of efficacy, treatment completion rates, and adverse events related to LTBI treatments for individuals with confirmed LTBI. These findings would support the current recommendations outlined in the 2020 WHO Consolidated Guidelines.To date, there have been three network meta-analyses (NMA) reporting on the efficacy11.This study was conducted in accordance with a PROSPERO-registered protocol, specifically identified as number CRD42020208880, and adhered to the reporting guidelines outlined in the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement extension for NMAA systematic search was performed on the MEDLINE database through PubMed and Scopus databases up until April of 2023. The study employed a predefined set of search terms encompassing ‘latent tuberculosis infection’, ‘tuberculosis infection’, ‘isoniazid’, ‘rifampicin’, ‘rifapentine’ and randomized controlled trails (RCTs). There were no restrictions imposed on the language or publication date. The search strategies are described in the appendix Table The inclusion criteria for this study comprised solely of RCTs involving participants diagnosed with LTBI through either a positive TST or an IGRA. The studies evaluated the efficacy of different treatment regimens for LTBI in terms of their ability to prevent the development of active TB, ensure treatment completion, and minimize adverse events. The treatment regimens under evaluation included 6H, 9H, 3HR, 3HP, 4R, in addition to placebo, a substance or treatment devoid of therapeutic efficacy or absence of treatment. The studies with insufficient data for pooling, despite our efforts to contact the authors, as well as the studies published in languages that were not translatable, were excluded from the analysis.The identified studies were reviewed based on the information presented in the title and abstract of each study by two independent reviewers (PY and DG). In cases where abstracts were insufficient in providing a conclusive determination, full articles were reviewed. Disagreement was resolved by reaching a consensus through discussion with the third reviewer (TA). The study's general information including the first author and publication year, the study characteristics including country, number of participants, follow up time in months, and participant characteristics including age, gender, comorbidity, method of LTBI diagnosis, treatment regimens and treatment duration, outcomes data including active TB disease, completion rates or adverse events were all collected, and the data for pooling were extracted into dichotomous outcomes.12. This tool consists of five domains: (1) the randomization process, (2) the intended interventions, (3) missing outcome information, (4) outcome measurement, and (5) selection of the reported result. Each item provides a response in the form of “yes” and “probably yes” or “no” and “probably no”. The “no information” response is only provided in cases where there is a lack of sufficient information. Each domain is classified into three categories: low risk of bias, high risk of bias, and some concern. An overall assessment is categorized as follows: (1) High risk of bias if at least one domain is determined to have a high risk, or if multiple domains are determined to have some concern. (2) Some concern if at least one domain is determined to have some concern, but none of other domains is determined to have a high risk of bias. (3) Low risk of bias if the result indicates a low risk of bias for all domains.The methodological quality of the included studies was assessed using RoB2, the second version of the Cochrane tool for assessing risk of bias in RCTsM. tuberculosis from acid-fast bacilli, sputum smear, clinical suspicion, and radiographic findings.The present study focused on active TB disease as the outcomes of interest. The definition of active TB disease varied across studies, depending on the acceptable standard of TB diagnostic used in each respective study. Some of the criteria used included positive results for 13. Grade 3 is considered a serious medical condition, while grade 4 is regarded as extremely dangerous and potentially life-threatening. The completion rates were determined by calculating the percentage of medication doses taken, with the requirement being that participants needed to have taken between 80% and 100% of the prescribed doses for each trial.Adverse events were classified as grade 3, 4, or 5, which included severe outcomes such as death or hepatotoxicity. These events were specifically related to treatment regimens or discontinuation caused by any adverse drug events. The term “grade” pertains to the severity of the adverse events, as defined by the Common Terminology Criteria for Adverse Events (CTCAE)14. If there was heterogeneity among the studies, the pooled RD was calculated using a random-effect model by DerSimonian-laird method15.A pairwise meta-analysis was performed to evaluate the efficacy of different regimens, focusing on three key outcomes: TB disease, adverse events, and treatment completion. The risk difference (RD), which represents a difference in risk of outcomes between two groups, with 95% confidence interval (CI) of each study was estimated. If there was no heterogeneity between studies, the results were pooled across all included studies using the inverse variance method2 statistic were further used to evaluate the heterogeneity among studies16. The Q test yields a p-value that signifies the likelihood of observing such variability due to random chance. A p-value below 0.1 typically indicates a notable degree of heterogeneity17. According to commonly used guidelines, Higgins’s I2 statistic is interpreted as follows: a range of 0%–25% indicates no heterogeneity, 25%–50% indicates moderate heterogeneity, 50%–75% indicates substantial heterogeneity, and 75%–100% indicates considerable heterogeneity15. Based on I2 index, when heterogeneity is present (I2 > 25%), it is recommended to apply a random effect model16. If the p-value from Cochrane’s Q test is less than 0.10 or if the Higgins’s I2 statistic is greater than 25%, it is inferred that there exists heterogeneity within the data, and a random effect model was applied. Alternatively, if the p value obtained from Cochrane’s Q test is greater than 0.10, or the Higgins’s I2 statistic is less than 25%, it is assumed that there is no evidence of heterogeneity, and a fixed effect model was applied.Heterogeneity was explored by the forest plot and statistical testing. The forest plot is a visual presentation that displays the point estimates of individual studies. If the point estimates are aligned on the same side as the vertical line, which represents the null effect, it is indicative of potential low heterogeneity. If the point estimates of each study are arranged on both sides of a vertical line, it could potentially indicate substantial heterogeneity. The Cochran’s Q test and Higgins’s Ip value is less than 0.05, it indicates the statistically significant presence of inconsistency. In such cases, a loop-specific approach was employed to identify the treatment arms and studies that contributed to the inconsistency. The source of inconsistency was explored. The comparison-adjusted funnel plot was utilized to address publication bias in NMA.A network meta-analysis was conducted to evaluate and compare the efficacy, completion rates, and occurrence of adverse events among various treatment regimens included in the studies. A two-stage approach was applied. In the initial stage, it is important to estimate the effect size (risk difference) and variance–covariance for dichotomous outcomes in each study. A multivariate random effect meta-analysis with consistency model was applied in the second stage to pool the risk difference data from the various studies. The various effect sizes were contrasted by estimating the relative treatment effects among different active treatment regimens. The probability of being the most effective treatment among all treatment regimens was determined using a rankogram and the surface under the cumulative ranking curve (SUCRA). The Y-axis represents the SUCRA value, a single numerical metric ranging from 0% to 100%, used to evaluate the likelihood of adverse events associated with different treatments in the NMA. The X-axis corresponds to the ranking, ranging from best to worst, based on the SUCRA values. Higher SUCRA values indicate a greater likelihood that a treatment is ranked at the top (best), with values closer to 100% indicating a higher probability. On the contrary, when SUCRA values are lower, approaching 0%, it indicates a higher likelihood that a treatment is positioned in the bottom rank (worst), indicating poorer performance. The consistency assumption was performed by applying the design-by-treatment interaction model to assess design inconsistency. If the 18. The risk difference ) was estimated through NMA. Afterwards, an incremental risk and benefit ratio (IRBR) was evaluated and simulated by the Monte Carlo simulation with 1000 replications, assuming normal distributions for both incremental risk and incremental benefit. All analyses were performed using STATA software package, version 16.0 .The risk–benefit assessment was performed to evaluate both the risk (adverse events) and benefits (TB prevention) simultaneously29, 11 studies32 and 10 studies33 were included in the NMA for efficacy, adverse events and treatment completion, respectively. The PRISMA flow diagram of electronic searching is shown in Fig. A total of 2400 records were identified from two aforementioned databases. Out of these, 72 articles were reviewed in full text as they were deemed potentially relevant. Out of the articles reviewed, 57 were excluded for various reasons. The majority of these exclusions were due to the fact that they did not pertain to the target population of interest (16 articles), did not address the outcomes of interest (14 articles), or did not involve the intervention of interest (10 articles). Additionally, 10 articles were excluded because they were not RCTs, and 7 articles were excluded for other reasons. In summary, a total of 15 studies were included in the present NMA. Of these studies, 11 studies23. The mode of administration was not reported in most studies (7 studies)32. Out of these, five studies reported administration through self-administered therapy (SAT) 33, while two studies reported administration through both directly observed therapy (DOT) and SAT26. Administration by DOT was reported in only one study30. Four studies were conducted on populations living with human immunodeficiency virus (HIV)28. Additionally, four other studies were conducted in prisoners30, immigrants21, kidney transplant patients23 and children20.The characteristics of included studies are summarized in Table 12. Overall, there were some concerns for nine studies33, high risk for two studies32, and low risk for four studies26. Details on grading of the quality of studies are shown in the appendix Figure The quality of study was evaluated using RoB2The network of eligible comparisons for efficacy, treatment completion, and adverse events outcomes is presented in Fig. Regarding the efficacy outcome, it was observed that 6H significantly reduced the risk of TB disease by 0.9%  compared to placebo. Compared to 3HR, there was a 0.3% increase in the risk of TB disease . No statistical heterogeneity was found in these pairwise comparisons, as shown in Table The NMA of 11 studies involving 33,262 participants assessed active TB disease of six treatment regimens for LTBI , followed by 3HP , 9H , 6H , and placebo . The ranking of treatments based on SUCRAs is displayed in the appendix Figure 34. This implies that studies with a longer follow-up duration should a greater number of cases for treatment comparisons. Subgroup analyses could not be performed due to the limited number of RCTs. The network's comparison-adjusted funnel plots displayed no evidence of asymmetry , compared to the use of 3HR. Compared to 4R, 9H resulted in a significant reduction of completion rate by 13.2% . Both exhibited moderate heterogeneity , followed by 3HR , placebo , 6H , and 9H . The ranking of treatments based on SUCRAs is shown in the appendix Figure A test for global inconsistency test was performed, and the results showed no evidence of inconsistency in the treatment network regarding treatment completion  compared to 3HR. Compared to 4R, 9H resulted in a 1.3% increase in adverse events . There was no evidence of statistical heterogeneity in these pairwise comparisons  and benefits (TB prevention). The NMA estimated the incremental risks and benefits of various treatment regimens  compared to 6H. The values for IRBRs were 0.004, − 11.040, − 7.905, − 0.097, and − 9.245 for placebo, 9H, 4R, 3HR and 3HP compared with 6H, respectively , 9H (− 9.8737), 4R (− 7.2616), and 3HR (− 0.0807) suggested that these treatment regimens had higher efficacy and fewer adverse events for those with confirmed LTBI in preventing TB compared to 6H, a standard treatment. In contrast, it was noticed that placebo had less benefit and less risk compared to 6H. This result can be used as the best available evidence to confirm that the benefits of 3HP, 9H, 4R, and 3HR outweigh the risks compared with 6H. This is also in line with the recommendations by the 2020 WHO Consolidated Guidelines for TB preventive treatment7, the National Tuberculosis Controllers Association (NTCA) and the 2020 Centers for Disease Control and Prevention (CDC)36 due to drug toxicity. Therefore, of all 31 studies in a previously published NMA9, 21 studies were not included due to our exclusion criteria and we added 5 new studies31, resulting in a total of 15 studies investigating efficacy, treatment completion and adverse events of LTBI treatment recommended by the guidelines among individuals with confirmed LTBI.Unlike two previously published NMA which included all participants receiving available LTBI regimens either with or without LTBI testing, this NMA included only those with confirmed LTBI to ensure that these participants should be given TB preventive therapy. Although one published NMA in 2017 included 31 studies consisting of individuals with confirmed LTBI, they analyzed only efficacy and treatment completion of all LTBI treatment including PZA and isoniazid for longer than 9 months which are not currently recommended by the 2020 WHO Consolidated Guidelines9. Nonetheless, two previously published NMA revealed that all LTBI regimens were more efficacious compared with placebo10. It should be highlighted that clinicians should consider not only the efficacy of LTBI regimens, but also the characteristics of individuals such as age, medication adherence, risk of adverse events, co-morbidity, and individual preferences should be taken into account7.In terms of TB prevention, compared with 6H, no significant differences in the efficacy of LTBI regimens were observed for 9H , 4R , 3HR  and 3HP . Similar results were also found in the study of Pease et al. indicating that 9H, 4R, 3HR, 3HP, and 6H did not have significant differences in efficacy10. In addition, there was no significant difference in completion rates between the 3HR and 6H groups. Although a poor adherence could lead to reduced benefit of TB prevention, there is no evidence to suggest that 3HR treatment is associated with reduced benefits. Several factors may also affect adherence, including the method of administration like SAT, DOT, drug tolerance, dose frequency (daily or weekly), and daily pill burden. The explanation for this distinction is likely owing to the fact that the 3HR regimen requires a higher daily pill intake and more frequent dosing compared to the 3 to 4-month regimens of 3HP and 4R.Moreover, it was observed that shorter regimens lasting for 3–4 months exhibited a higher rate of completion. We found that the completion rates of 3HP  and 4R  were significantly increased by 12% compared with 6H. This is consistent with the results from two previously published NMA, showing that the regimens of 3–4 months had a higher chance of regimen completion7. This study was the first NMA that compared the occurrence of adverse events among participants with confirmed LTBI who received LTBI regimens. It was discovered that the use of 3HP, 4R, and 9H resulted in a statistically significant decrease in adverse events by 4.53% , 4.39%  and 3.77%  compared to 6H, respectively. However, no significant differences in adverse events observed between placebo  and 3HR  compared to 6H. Our findings were different from the study of Assefa et al. published in 2023 which revealed that adverse events were occurred in 3HR treatment group and the risk of hepatotoxicity was significantly higher in 9H followed by 6H compared to placebo, while the other previous NMA did not have data available on the hepatotoxicity of isoniazid and rifampicin regimens8.It should be emphasized that the risk of adverse events should be carefully monitored together with the potential benefit of LTBI regimens37.Our results indicated that the administration of 3HP, 4R and 9H led to a significant reduction in adverse events as compared to 6H. Despite a longer treatment duration, 9H significantly reduced the occurrence of adverse events. It can be explained that LTBI patients receiving 9H tended to have a lower risk of adverse events than those receiving 6H. It is worth noting that the latter group consisted most patients who were also HIV-positive. Given that HIV infection is one of the risk factors attributable to idiosyncratic drug-induced liver injury (DILI), people living with HIV receiving multiple treatments for both HIV and opportunistic infection treatments might suffer from toxicity related to the interaction of drug–drug and drug–disease38. In addition to these risk differences, number needed to treat (NNT) to prevent one event calculated as the inverse of the absolute risk difference, has been applied to assess clinical significance39. It is also highlighted that the established threshold exists for NNT which are generally considered as clinically significant when NNT is less than 50 for treatment interventions39. Consequently, our results indicated that 6H significantly decreased the risk of TB disease by 0.9% (NNT = 11) compared to placebo. Compared to 6H, the completion rates of 3HP and 4R were significantly increased by 12% (NNT = 8), while the adverse events of 3HP, 4R, and 9H significantly decreased by 4.53% (NNT = 22), 4.39% (NNT = 23) and 3.77% (NNT = 27), respectively. Given that the reported NNT values meet such threshold, our findings can shed light on the practical implications of these results in clinical practice.However, our reported risk differences seem relatively small in magnitude even though these differences were statistically significant. It is worth noting that the small extent of these risk differences should be interpreted with the consideration of their clinical significance which will determine whether the results of the RCTs are likely to reflect the impact on real clinical practice40. For that reason, we excluded studies containing regimens with PZA to confirm that included studies were comparable interventions. Nevertheless, this could result in less included studies which could lead to potential bias. Therefore, additional RCTs are required to validate this finding and enhance statistical power in future studies. Secondly, we found that the studies included in our review had varying lengths of follow-up time and measured efficacy outcomes related to TB prevention, which may have long-term benefits. However, due to these differences in follow-up time, our review was unable to account for them. Therefore, future studies require more evidences for adverse event outcomes, considering different follow-up time for efficacy outcome and incorporating other factors such as HIV positive patients receiving antiretroviral therapy. Additionally, the recent evidence for a shorter duration of treatment, a 1-month regimen of rifapentine plus isoniazid should be included.It was noteworthy to address the limitations in our review. Firstly, it is important to note that the standard treatment regimens are different, and standard practice is changeable over time. According to published reports on the toxicity profile of PZA, it is recommended that PZA should be restrictedly usedIn conclusion, our risk–benefit assessment suggests that 9H, 4R, 3HR and 3HP had higher efficacy and fewer adverse events than 6H, the standard treatment. This information could be applied to support the current clinical practice for TB preventive treatment. Moreover, this comprehensive review summarized the effect of LTBI treatment regimens on efficacy, completion rate, and adverse events. According to the NMA, there was no significant difference in the prevention of active TB among the various active treatment options. Additionally, it was found that treatment regimens 3HP and 4R had a higher completion rate. Furthermore, regimens 3HP, 4R, and 9H showed a decrease in the occurrence of adverse events. Considering on efficacy, completion rate, adverse events, and risk–benefit assessment results, the treatment regimens 3HP and 4R would be highly recommended for individuals with confirmed LTBI. This information could be used to support the recommendation from the 2020 WHO Consolidated Guidelines and assist clinician and public health program management for TB preventive treatment.Supplementary Information 1.Supplementary Information 2.Supplementary Information 3.


ADH1B+ CAF enrichment is associated with distinct ECM and immune cell signatures in early-stage LUADs. Also, we found a positive correlation between the gene expression of ADH1B+ CAF markers in early-stage LUADs and better survival. We performed TCGA dataset analysis to validate our findings. Identified associations can be used for the development of the predictive model of LUAD aggressiveness and novel therapeutic approaches.Lung adenocarcinoma (LUAD) is the predominant type of lung cancer in the U.S. and exhibits a broad variety of behaviors ranging from indolent to aggressive. Identification of the biological determinants of LUAD behavior at early stages can improve existing diagnostic and treatment strategies. Extracellular matrix (ECM) remodeling and cancer-associated fibroblasts play a crucial role in the regulation of cancer aggressiveness and there is a growing need to investigate their role in the determination of LUAD behavior at early stages. We analyzed tissue samples isolated from patients with LUAD at early stages and used imaging-based biomarkers to predict LUAD behavior. Single-cell RNA sequencing and histological assessment showed that aggressive LUADs are characterized by a decreased number of ADH1B The high death rate of lung cancer is mostly associated with its late diagnosis, and low-dose chest CT screening helps to detect lesions early and significantly reduces lung cancer mortality3. Lung adenocarcinoma (LUAD) is of particular interest because it is the predominant histological type of lung cancer within the U.S. and is rapidly growing in other countries, and it exhibits a wide spectrum of behavior that can be indolent or very aggressive characterized by rapid tumor growth, invasion of healthy tissue and metastasis4. Thus, patients with indolent LUAD at early stages often undergo overdiagnosis and overtreatment, whereas individuals with aggressive tumors require urgent therapeutic procedures. Selection and utilization of the correct treatment strategy requires novel methods for the prediction of LUAD behavior and prognosis. For this purpose, it is necessary to identify and better understand the biological determinants of LUAD aggressiveness.Lung cancer is the leading cause of cancer death in the United States and worldwide, killing 1.8 million individuals every year6. Nevertheless, existing predictive models are based on the biology of the tumor cells and ignore other components of the tumor microenvironment which can be specific to LUAD. Cancer behavior is associated with extracellular matrix deposition and remodeling, and cancer-associated fibroblasts (CAFs) play a critical role in these processes8.Biological mechanisms associated with LUAD behavior are still poorly characterized. Several reports have demonstrated that LUAD behavior is associated with the histological subtype of the tumor, anti-cancer immune response, mutational burden, age, and smoking status of the patients7. Beyond the functions associated with the ECM, CAFs are involved in a number of different processes: modulation of immune response, tumor cell collective migration and invasion, angiogenesis, and metabolic reprogramming14. For instance, CAFs can produce cytokines that can modulate anti-cancer immune responses15. However, it is still not clear if CAFs act only as a pro-tumorigenic factor or, depending on the context, they can play an anti-tumorigenic role14. Context-dependent involvement of CAFs in determination of tumor progression can be used for tumor behavior prediction. Recent progress in single-cell technologies gives an opportunity to perform a more detailed and precise analysis of CAFs in the tumor microenvironment.CAF populations in the tumor microenvironment are diverse and characterized by different origins, spatial distribution, and markers16 to perform stratification of early-stage LUAD patients based on predicted indolent or aggressive behavior of the tumors. We performed the analysis of the tumor samples using single-cell RNA sequence (scRNAseq) and histological assessment. We parsed apart separate subpopulations of CAFs in the tumor microenvironment and identified the association between the ADH1B+ CAF enrichment of LUAD at early stages and predicted tumor behavior. Our findings were confirmed through the analysis of the TCGA dataset from LUAD patients.Here, we used an orthogonal, previously validated imaging-based biomarkersThe experimental protocols were approved by the Vanderbilt University Medical Center Institutional Review Board (IRB protocol #000616). All patients involved in the research provided informed consent. Lung adenocarcinoma tissue was isolated from 16 patients during tumor resection at the Vanderbilt University Medical Center . SILA ranges from 0 to 1 and is determined by 9 radiologically distinct exemplars16. Using a variety of geometrical and textural features of lung tumors, the CANARY is able to distinguish in situ and invasive carcinomas, predict the invasiveness of the tumor, as well as LUAD patient survival and mortality at the early stage of progression. We set a SILA threshold at 0.5 to distinguish patients with promising and poor predicted prognoses. The group with a SILA score lower than 0.5 was assigned as indolent, and the patients with a SILA score higher than 0.5 were assigned as aggressive. The indolent group was composed of 9 patients and the aggressive group was composed of 7 patients  software as described previouslyThe tumor tissue samples were placed in cold PBS immediately after the surgery and divided into two parts for histological analysis and scRNAseq . Tissue samples for histological assessment have been examined by pathologist and placed in a 10% neutral buffered formalin for 24 h at room temperature for fixation. The samples for scRNAseq have been dissociated into a single-cell suspension within one hour. To prepare the digestive media, 1 mg/ml Collagenase I  and 1 mg/ml of DNAse I  were mixed in DMEM media . Minced tumor tissue was incubated in the media in a shaking water bath for 1 h at 37 °C. After incubation, tumor tissue was disrupted using a syringe pestle and filtered through 70 um strainers to remove debris. The suspension was additionally filtered using a 30 um strainers. Single-cell suspensions were cryopreserved for long-term storage. Cells were counted, and their viability was assessed before and after cryopreservation.19. Cells with either less than 250 or more than 4000 genes detected were filtered out. Additionally, cells with a percentage of mitochondrial genes higher than 17% were excluded from the analysis. Cell populations were annotated in accordance with gene expression patterns that have been demonstrated in prior literature. For dimensionality reduction, we used the Uniform Manifold Approximation and Projection algorithm (UMAP)20. The Leiden algorithm was used for the clustering of cell populations21. In the samples isolated from patients 3, 6, and 7, no stromal cells were identified. The composition of patient samples' cells could have been affected by procedures related to sample isolation, tumor tissue dissociation, or cryopreservation.Cryopreserved single-cell suspensions prepared from isolated LUAD tissues were used for scRNAseq. Dead cells were removed using a MACS Dead Cell Removal Kit . From each sample, 5000–10,000 cells were used for scRNAseq using a 10× Genomics platform . Library preparation was performed in accordance with the manufacturer’s protocol. The libraries were sequenced using a NovaSeq 6000 with 150 bp paired reads. Feature matrices preparation was performed using 10× Genomics Cell Ranger software . The library was aligned to the GRCh38 reference genome. The analysis of the scRNAseq dataset was completed using a Python 3 package, ScanPy+ CAFs. Epithelial/tumor cells were detected using anti-PanCK-Alexa Fluor 488 antibodies , and ADH1B+ CAFs were visualized using rabbit anti-human antibodies . Nuclei were located using Gold Antifade Mountant with DAPI . For visualization of collagen in the tissue samples we used CHP–Collagen Hybridizing Peptide  staining labeled with Cy3 dye. Tissue images were obtained using the Keyence BZ-X710 microscope  using a 10× objective with a Texas Red filter , a GFP filter , and a DAPI filter . Tissue image analysis was performed using ImageJ22 and QuPath23 software. LUAD histological subtypes were defined by a pathologist. When multiple subtypes were identified in one tissue one subtype was assigned as predominant based on the area of tissue occupied. To estimate collagen deposition and assess cell numbers, we analyzed at least 10 areas for each sample.After fixation tissue samples have been embedded in paraffin. Next, 5–7 um tissue sections were prepared. The tissue samples were examined by a pathologist to determine the histological subtypes of lung adenocarcinomas. Hematoxylin and eosin (H&E) and multiplex immunofluorescent (MxIF) staining were used for tissue analysis. MxIF staining was used to identify tumor cells and ADH1B24, “limma”25, and “edgeR”28) to download and analyze a TCGA dataset. The data of 419 patients with LUADs at stages I and II  were assessed. The cohort was composed of 323 Caucasians, 6 Asians, and 43 African Americans, and 47 samples with missing racial and ethnic information. All other parameters such as sex, age, treatment, histological type, and recurrence, were not taken into account. Specifically for Fig. We used R programming language and appropriate packages  curves, and Cox models. In addition, stratified Kaplan–Meier plots were generated using available web Kaplan–Meier plotter30. The tool uses information downloaded from The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO), and European Genome-Phenome Archive (EGA). LUAD samples at stage I were analyzed using this tool. The dataset was not adjusted to specific criteria such as sex, age, treatment, recurrence etc.Survival analysis of the downloaded TCGA dataset has been performed using “survival” packagehttps://cibersortx.stanford.edu). The algorithm implements linear support vector regression to calculate the relative levels of cells. Signature matrix for 3 CAF subpopulations were generated using our scRNAseq dataset. For estimation of immune cell enrichment score we used predefined signature matrix.To calculate cell enrichment score for bulk RNAseq data (TCGA dataset) we utilized the CIBERSORT algorithm (https://www.graphpad.com), Python 3 (https://www.python.org) and R 4.2.1 (https://www.r-project.org). One-way ANOVA followed by Dunnett’s procedure was used for multiple comparisons between groups. A two-sided comparison was performed using a two-sample t test or a Wilcoxon Rank-Sum test as appropriate. For correlation analysis, we used Pearson correlation coefficient. The Kaplan–Meier method was used to visualize survival plots and a log rank test was used for an estimation of statistical significance. All tests were considered statistically significant at a two-sided 5% level.Statistical analyses and plot generation were performed using GraphPad Prism 9 , endothelial cells , stromal cells , mast cells , myeloid cells , CD8+ T cells , NK cells , B-cells (CD79A+), CD4+ T-cells , and plasma cells   was used to perform a comprehensive analysis of the CAF population in the tumor microenvironment. This analysis identified 43,207 cells with distinct transcriptomes. 21,140 cells (48.93%) were obtained from indolent LUADs and 22,067 (51.07%) were from aggressive LUADs. Using graph-based uniform manifold approximation and projection algorithm (UMAP)+ACTA2+ CAFs. Cluster 1 was identified as ADH1B+ CAFs. Since normal fibroblasts also express ADH1B, we employed another marker of normal fibroblasts, CD10 (MME), to corroborate that the identified ADH1B+ population consists of CAFs. Our findings revealed that the ADH1B+ cluster lacks or demonstrates very low CD10 expression, further confirming that the cells within this cluster are indeed CAFs and not normal fibroblasts  from indolent LUADs and 187 cells (40.4%) from aggressive carcinomas. The cluster of ADH1B+ cells was formed by 234 (71.8%) and 92 (28.2%) CAFs from the indolent and aggressive tumors, respectively. The group of CTHRC1+FAP− CAFs consists of 153 cells (56.2%) from indolent and 119 cells (43.8%) from aggressive LUADs. The comparison of the average proportions between different types of CAFs revealed that aggressive LUADs are characterized by a decreased proportion of cells that form the ADH1B+ CAF cluster (p = 0.032)  enrichment analysis indicates that CAFs from the FAPing Fig. B. Indeeding Fig. C. In addway Fig. D.+ subpopulation also has been involved in the deposition and remodeling of ECM structures , A2M and MFAP4 as top marker genes of this subpopulation of cells  in comparison to the aggressive group, supporting the results obtained using scRNAseq , A2M, MFAP4, SEPP1 (SELENOP), and DCN   for this purpose. Patients were stratified into two groups based on the expression of several marker genes (high and low): ADH1B, CFD (DF), A2M, SEPP1 (SELENOP), and DCN. The median expression was used as separation point. Patients with high expression of ADH1B , CFD (DF) , SEPP1 (SELENOP) , DCN , and A2M  demonstrated better survival at stage I of LUAD , SELENOP (SEPP1), DCN, A2M and MFAP4. The analysis of the overall survival of LUAD patients demonstrated better survival of the patients with higher expression of these marker genes. The analysis of bulk RNAseq dataset obtained from TCGA demonstrated that high expression of the ADH1B+ CAF markers is associated with lower enrichment of infiltrating immune cells, and reduced expression of different types of collagens and upregulated expression of DCN, LAMA2, and ELN that is consistent with the results obtained using scRNAseq and histological assessment of LUAD samples from our cohort. The results of our research show that ADH1B+ CAFs correlate with LUAD behavior and can be used for the prediction of tumor aggressiveness at an early stage.A better understanding of the biological determinants associated with LUAD aggressiveness at the early stages of tumor development could significantly improve existing predictive models and reduce mortality18. The stratification was based on the SILA score—a computerized scoring system designed for predicting LUAD behavior and estimating patient survival. The robustness of this method has been demonstrated in several studies33. Nonetheless, a better understanding of the biological processes associated with LUAD behavior at early stages could considerably enhance existing prognostic methods. In the tumor microenvironment CAFs participate in a number of biological processes including ECM deposition and remodeling, regulation of anti-cancer immune response, angiogenesis, and tumor cell invasion11. Despite the active investigation of CAF function, it is still not clear if CAFs act only as a pro-tumorigenic factor or, depending on the context, if they can play an anti-tumorigenic role14. We have identified 3 distinct CAFs subpopulations in early stage LUAD tumors: FAP+ACTA2+, ADH1B+, and CTHRC1+FAP-. This indicates CAF diversity even at early stages of LUAD progression. Moreover, altered ratios of these CAF subtypes in the indolent and aggressive LUADs and in LUADs characterized by different histological subtypes may indicate different mechanisms of CAF activation, transition between CAF subtypes, and involvement in the determination of LUAD behavior. Grout et al.31 have demonstrated that enrichment of LUAD tissue with different subtypes of CAFs is determined by tumor stage and histological subtypes. Their work showed that ADH1B+ CAFs were enriched in early stage LUADs of the papillary subtype, whereas FAP+ CAFs dominated in late stage tumors of the solid histological subtype31. Our cohort mainly comprises samples characterized by acinar and solid histological patterns. We have identified that FAP+ACTA2+ CAFs dominate in this LUAD tissue, supporting the findings demonstrated by Grout and colleagues. Interestingly, we did not observe a significant difference in proportion between FAP+ACTA2+ and CTHRC1+FAP- CAFs in the indolent and aggressive groups. This can be associated with the early stage of LUADs where these types of CAFs may not play a critical role. On the other hand, revealed disproportion in ADH1B+ CAFs could indicate their role as one of the determinants of LUAD behavior at early stages.For patient stratification, we have used CANARY software as described previously+ subpopulation displayed low activity of signaling pathways including TGFβ, WNT, hypoxia, EGFR, TNFa, and VEGF, which implies low level of activation of these fibroblasts and places them in an intermediate state between normal fibroblasts and activated CAFs. In fact, Grout et al.31 have assumed that the ADH1B+ subpopulation of CAFs acts as a transient form between normal fibroblasts and FAP+aSMA+ CAFs. Identified similarity in gene expression between all 3 subtypes of CAFs may indicate that they could be transient forms of each other. However, this hypothesis requires further exploration.We have demonstrated that identified CAF subpopulations are characterized by the different activity of several signaling pathways. This may be related to either difference between distinct phenotypes or different mechanisms of CAF activation. The ADH1B+ CAFs participate in matrix deposition as well, but these CAFs display higher expression of DCN, LAMA2, and ELN and lower expression of different types of collagens in comparison to FAP+ACTA2+ CAFs. Interestingly, high expression of DCN, LAMA2, and ELN in lung cancer is associated with better survival37. Additionally, CAFs participate in the modulation of the anti-cancer immune response15. Beyond ECM related functions, ADH1B+ CAFs were characterized by increased expression of cytokines such as CXCL12, and IL33, which indicates these CAFs can be involved in immune-modulatory processes. The role of these cytokines is controversial, and it is not clear how exactly they affect the tumor microenvironment of LUADs, especially at early stages. However, several reports have shown that expression of IL33 in the tumor microenvironment correlates with better prognosis and can be associated with activation of NK cells and cytotoxic T cells40. Our results indicate that aggressive LUADs are characterized by higher immune cell enrichment including regulatory T cells and neutrophils that are associated with poor survival. In LUADs, several reports discovered CAFs that were characterized by an increased expression of different cytokines and were involved in immune response modulation processes. For example, Xing et al.41 identified CFD+CXCL14+ CAFs that uniquely expressed CXCL12 and CXCL14. Grout et al.31 identified that ADH1B+ CAFs had a high expression of IL34, CSF1, CCL19, CCL21, and CXCL12. Interestingly, we identified upregulation of CFD, C3 and C7 in ADH1B+ CAFs that are part of complement cascade. Complement has a pleotropic role in the tumor microenvironment. However, several reports indicated that complement activity can be related to the better prognosis43.Another important question is how exactly identified CAF subpopulations can affect the behavior of LUADs and in what biological processes are they involved in the tumor microenvironment? GO enrichment analysis showed that ADH1B+ CAFs can be used not only for better understanding of mechanisms associated with LUAD aggressiveness but also for the development of novel predictive models. It is necessary to discover and validate a set of markers associated with ADH1B+ CAFs that could be identified by different methods on the transcriptomic, proteomic, and cellular levels. We have defined the top 5 marker genes , SEPP1 (SELENOP), DCN, and A2M) that could be used for the identification of ADH1B+ CAFs in tumor tissues. An improved overall survival of the LUAD patients with high expression of these genes confirmed our hypothesis regarding the involvement of ADH1B+ CAFs in determining LUAD behavior.The identified subpopulation of ADH1BOur work has several limitations. First, it is possible that the observed intratumoral cellular heterogeneity is associated with the accidental resection of healthy lung tissue due to the small size of the early-stage tumors. Second, the possible biological mechanisms that determine the presence of different CAFs subpopulations in the LUAD tissue were not investigated. Third is the small size of the patient cohort (9 indolent vs. 7 aggressive LUADs). We combined different approaches to overcome these limitations.+ CAFs deficiency in early-stage LUADs is associated with tumor aggressive behavior. ADH1B+ CAFs could potentially be used as a marker to predict LUAD behavior and for a better understanding of the biological mechanisms associated with LUAD aggressiveness. We highly suggest further investigation of this subpopulation of CAFs to better understand their origin and function in the tumor microenvironment.In conclusion, we showed that ADH1BSupplementary Information.