Thymus schimperi), DA8 (75 g flaxseed and 800 mg Dodonaea angustifolia) and CD8 (75 g flaxseed and 800 mg Curcuma domestica) extract per kg diet. Feeding on CD8 and VE8 in raw and TS8, CD8 and VE8 diets in cooked breast muscle increased (p < 0.05) the C22:6n − 3 (DHA) and C20:5n − 3 (EPA) contents compared to the FS diet. Feeding FS increased (p < 0.05) the malondialdehyde (MDA) content in breast muscle, whereas TS8 in cooked and raw and CD8 and DA8 diets in raw breast muscle decreased it (p < 0.05). No added benefit was observed in feeding VE8 over plant extracts in terms of improving fatty acid composition and lipid health indices and reducing lipid oxidation in breast meat.Adding flaxseed was found to decrease oxidative stability in feed and increase the antioxidant needs of chicken. This has also been associated with a decrease in the nutritional value and oxidative stability of meat if sufficient dietary antioxidants are not included. Furthermore, dietary flaxseed has been explored in fast-growing chickens as such studies are limited with slow-growing chickens. Thus, this study aimed to evaluate the effects of feeding plant polyphenol extracts as an antioxidant alongside flaxseed on fatty acid content, oxidative stability, and lipid health indices in breast muscle of slow-growing Sasso T451A dual-purpose chicken. A total of 126 chickens assigned to six groups (seven replicates of three) were fed on NC (control and no antioxidants), FS (75 g flaxseed and no antioxidants), VE8 (75 g flaxseed and 800 mg vitamin E), TS8 (75 g flaxseed and 800 mg  However, the marine ecosystem is under constant pressure raising the issue of long-term sustainability of the sector [n − 3 LC-PUFA sources [n − 3 LC-PUFAs.Most poultry industries’ primary focus is on increasing meat and egg production. However, it should also focus on the production of healthy eggs and meat for improved consumer health . One impe sector . Thus, i sources . Chicken sources  and is m sources . Hence, Linum usitatissimum L.) has been included in chicken diets as an alpha-linolenic acid (ALA) precursor to enrich egg and meat with n − 3 LC- PUFA [Flaxseed/linseed  has been utilized to treat lymphatic swelling and burn healing [Thymus schimperi (T. schimperi) has been commonly used as a traditional herbal tea drink. It is believed to treat cough and liver diseases [Curcuma domestica (C. domestica), which has antioxidant properties, is a common ingredient in every Ethiopian kitchen for the preparation of traditional sauces and had several health benefits [The diversity of medicinal plants available in Ethiopia could offer the opportunity to extract effective antioxidants to inhibit oxidative stress . Dodonae healing  and was  healing . Thymus diseases , and to diseases . Additiobenefits ,31,32.n − 3 PUFA source in chicken diets has been tested widely in fast-growing commercial meat [D. angustifolia, T. schimperi, and C. domestica extracts along with dietary flaxseed could improve the muscle fatty acid composition, oxidative stability, and lipid health indices of SassoT451A slow-growing dual-purpose chicken. More specifically, the study aimed to determine the effects of the supplementation of T. schimperi, D. angustifolia, and C. domestica extracts as polyphenol antioxidants and a higher dose of vitamin E, along with flaxseed as an ALA precursor in the diets of slow-growing Sasso chicken on fatty acid content, oxidative stability, and lipid health indices in raw and cooked breast meat.Up to now, the effect of the inclusion of flaxseed as an ial meat ,33,34 anial meat ,36. Studial meat ,38. In tThe current study protocols were approved by the Institutional Animal Care and Use Committee (IACUC2019-17.2) of the International Livestock Research Institute (ILRI), following the Sasso dual-purpose chicken management guide obtained from Hendrix Genetics  . At 35 wD. angustifolia extract/kg, TS8: 75 g flaxseed + 800 mg T. schimperi extract/kg; and CD8: 75 g flaxseed + 800 mg C. domestica extract/kg diets for 8 weeks. Hens were provided with 165 g feed/day and water ad libitum. They were vaccinated according to the recommended schedule and provided with 15 h lighting during laying. The chemical composition, nutrient levels, and fatty acid composition of the diets are provided in The chickens were fed on normal control (NC): 0 g flaxseed + no antioxidants sources, FS: 75 g flaxseed + no added antioxidant sources, VE8: 75 g flaxseed + 800 mg α-tocopherol/kg; DA8: 75 g flaxseed + 800 mg n − 3, Larodan, Limhamn, Sweden) and QuantiChrom TBARS Assay kit  and Conjugated linoleic acid . ABTS , DPPH, , Gallic acid, Trolox , Fatty acid methyl esters (FAMEs), Adrenic Acid (C22:4n − 6) were purchased from Sigma-Aldrich, Germany.Chemicals: Vitamin E , Methanol , Ascorbic acid , Stearidonic acid , Filter paper , Incubator Shaker , Rotary Evaporator , Drying oven, , Freeze dryer , UV-Vis spectrophotometer , Sarstedt tubes , Digital Kitchen thermometer , Centrifuge , Lab mill , Bulk beads . Microwave Plasma-Atomic Emission Spectrometer , Homogenizer , Pyrex tubes , CP-Sil 88 CB column , PerkinElmer gas chromatograph CLARUS 680 , 96-well-plate of the plate reader , Food saver vacuum bag  and Water bath .The dry matter and the total nitrogen were determined according to , with MeThe leaves, rhizomes of tested plants, and diet samples were powdered using a lab mill. Fifty milliliters of methanol was added to 5 g of powdered plant and feed samples in a conical extraction flask and shaken for 24 h at 25 °C. The solution was then filtered through a paper filter 3× to recover the extracts fully. Methanol was removed by a rotary evaporator followed by the concentration of the filtrate at 40 °C for 10 min using a drying oven. Methanol was completely removed using a freeze dryer, and the yield was calculated as per  and keptT. schimperi diluted to 1000 µL with a final concentration of 0.60 mg/mL. Similarly, 150 µL from 1.5 mg/mL for each D. angustifolia and C. domestica was taken in test tubes and diluted to 1000 µL, giving a final concentration of 0.225 mg/mL. Subsequently, 3.16 mL distilled water and 200 µL Folin-Ciocaltu reagent were added to each test tube containing plant and diet extracts. As a blank, methanol was added in place of the samples. After 8 min incubation at room temperature, 600 µL sodium carbonate (7.5%) solution was added and the solution was incubated at 40 °C for 30 min in the dark. Absorbance was recorded using a UV-Vis spectrophotometer at 765 nm against the blank. Gallic acid standard curves were developed for diet and plant extract samples. All determinations were conducted in triplicate. The equations obtained from the respective GA standard curves were used to calculate the total phenolic content as Gallic acid equivalent (GAE) per gram dry weight of the extracts and diets. The total phenolic content (TPC) of the plant extracts and diets was determined as per , with slT. schimperi, D. angustifolia, and C. domestica, a concentration of 10, 50, 90, 130, and 170 to 210 μg/mL were prepared from an initial concentration of 1000 µg/mL. Then, 4 mL of 0.04 mg/mL of DPPH radical was added to tubes containing diet extracts at 2.5 mg/mL and plant extracts at different concentrations. One milliliter of methanol and 4 ml DPPH solution were prepared as blank. Reference curves were established from concentrations of ascorbic acid for diets and plant extracts. Test tubes were incubated for 1 hr at room temperature in the dark and absorbance was read at 517 nm using a UV-Vis spectrophotometer. All determinations were completed in triplicate. The capacities of plant and diet extract to scavenge DPPH radical as µM of ascorbic acid equivalent (AAE) were calculated from the ascorbic acid standard curve. In addition, the percentage of DPPH radical inhibition by various doses of the plant extracts were calculated by comparing the absorbance of the sample and the blank. The concentration plant extracts to inhibit 50% DPPH radical (IC50) values were generated by using the “drc package’ in R [The DPPH  radical scavenging assay was carried out according to , with slge’ in R .v/v until the absorbance value remained between 0.800 to 0.100 at 734 nm. Aliquots of 10 µL, from each diet extract with 50 mg/mL initial concentration, were diluted to 1000 µL to bring the final concentration to 0.5 mg/mL. For extracts of T. schimperi, D. angustifolia, and C. domestica a concentration of 5, 20, 35, 50, 65, and 80 μg/mL were prepared from an initial concentration of 1000 μg/mL. Subsequently, 3 mL of diluted ABTS radical was added to the test tubes containing 0.5 mg/mL diet and plant extracts at various concentrations and incubated for 5 min at 30 °C. Trolox was used as a standard antioxidant in similar concentrations to the plant extracts. As a blank, a mixture of 3 mL ABTS radical solution and 1 mL methanol was used. Absorbance was measured at 734 nm using a UV-Vis spectrophotometer. Reference curves were established from different concentrations of Trolox. The antioxidant capacities of the plant and diet extracts to scavenge ABTS radical as µM Trolox equivalent antioxidant capacity (TEAC) were calculated using the equations established from Trolox calibration curves. The plant extract’s capacity to scavenge ABTS radical as a percentage of inhibition was calculated after subtracting the absorbance of the sample from the blank. The specific dose for each plant extract to inhibit 50% ABTS radical (IC50) was determined using the “drc package” in R [The ABTS  assay was applied to measure the antioxidant capacity of plant extracts and test diets as described by . The ABTge” in R .At the end of the 8-week feeding trial, two out of three hens per replicate, (14 per treatment) were killed by cervical dislocation and bleeding . ScaldinChicken diets2CO3 solution was added and vortexed. All solvents used for feed lipid extraction contained 0.005% (w/v) of t-butylhydroxytoluene (BHT) to prevent oxidation of PUFAs. Next, the solutions were centrifuged at 4 °C, 1200× g for 5 min and finally the fatty acid methyl esters (FAMEs) were extracted two times each with 2 mL of n-hexane. After drying (1 g Na2SO4) and cleaning of the organic phase, if necessary with activated charcoal, the extracts were filtrated and evaporated using a vacuum centrifuge at 438 g at 30 °C for 30 min. Finally, the extracts were stored at −18 °C until GC analysis [The frozen diet samples were finely ground using liquid nitrogen in a mortar with a pestle. For extraction and direct fatty acid methylation of diet samples, a modified method from  was usedanalysis .Chicken breast muscle w/v) of t-butylhydroxytoluene (BHT) to prevent the oxidation of PUFAs.The frozen breast muscle samples  were cut into small pieces and homogenized using a lab mill. For lipid extraction, approximately 1 g of breast muscle was weighed. Each Precellys-tube contained 20 pieces of 2.8 mm bulk beads and 2 pieces of 5 mm bulk beads. After the addition of 3 mL methanol and nonadeconoic acid (19:0) as an internal standard, the extracts (in duplicate) were homogenized 3 times at 25 s intervals at 4 °C and 6500 rpm using a homogenizer . The hom2 solution. The organic phase was separated and dried with a mixture of Na2SO4 and K2CO3 , and the solvent was subsequently removed using a vacuum centrifuge at 438 g/min, 30 °C, 30 min. The lipid extracts were redissolved in 300 μL of toluene, and a 25 mg aliquot was used for methyl ester preparation [n-hexane and stored at −18 °C until high-resolution gas chromatography (HR-GC) analysis.After filtration, the lipid extracts of muscle samples were stored at 5 °C for 18 h in the dark and subsequently washed with a 0.02% CaClparation . Briefly−1 while the split ratio was 1:20, with the injector and detector set at 260 °C and 280 °C, respectively. The GC oven temperature program was 150 °C for 5 min; heating rate of 2 °C/min until 200 °C and kept for 10 min; heating rate of 1 °C/min until 225 °C and kept for 20 min. For the calibration of the reference standard mixture “Sigma FAME,” the methyl ester of C18:1cis-11, C22:5n − 3, and C18:2cis-9, trans-11, C22:4n − 6, and C18:4n − 3 were used. The five-point calibration of single fatty acids ranged between 16 and 415 µg/mL and was assessed after GC analysis of five samples. Fatty acids were displayed as a concentration in mg/100 g of tissue.The fatty acid analysis of all sample extracts was performed using capillary high-resolution gas chromatography (HR-GC) with a CP-Sil 88 CB column. The GC column was installed in a PerkinElmer gas chromatograph CLARUS 680 with a flame ionization detector and split injection as described earlier . BrieflyThe frozen breast muscle samples  were cut into small pieces and homogenized using a lab mill. For the analysis of oxidative stability, approximately 400 mg of muscle sample was weighed in a tube. Each 7 mL Precellys-tube contained 5 pieces of 2.8 mm bulk beads and 1 piece of 5 mm bulk beads. The sample preparation and measurement of oxidative stability in breast muscle samples was performed according to the guidelines of the assay QuantiChrom TBARS Assay kit. After the addition of 2 mL of ice-cold phosphate-buffered saline (PBS) solution (200 mL and 133 µL BHT), the extracts (in triplicate) were homogenized 2 times at 10 s intervals at 4 °C and 6500 rpm using a homogenizer. g, and 200 µL of thiobarbituric acid (TBA) was added to 200 µL sample extract vortexed and incubated for 60 min at 100 °C. After cooling to room temperature, 100 µL of the sample extracts were transferred into the 96-well-plate of the plate reader. Then, the colour intensity (OD) was measured at 535 nm. The standard solutions of malondialdehyde (MDA) were prepared at a concentration range from 0.0 to 1.5 µM MDA and the colour intensity was measured using the same procedure as for the sample solutions. The concentrations of thiobarbituric acid reactive substances (TBARS) were calculated using the MDA standard calibration. Finally, the TBARS concentration was expressed in µg MDA/g of breast muscle.Then 200 µL of ice-cold 10% trichloroacetic acid (TCA) solution was added and incubated for 5 min on ice. After that, the sample extracts were centrifuged for 5 min at 14,000 n − 6/n − 3 PUFA ratio, saturation indices as saturated and unsaturated fatty acids ratio (s/p), atherogenic index (AI), thrombogenic index (TI) and hypocholesterolemic/hypercholesterolemic (h/H) ratio were estimated. The AI, n − 6/n − 3 PUFA, s/p ratios, the total sum of fatty hypercholesterolemic fatty acids (HFA), and desirable fatty acids (DFA) were calculated as per the below Equations (1)–(4) [PUFA—polyunsaturated fatty acids; MUFA—monounsaturated fatty acids; UFA—unsaturated fatty acids.The potential health contribution of lipids can be calculated by considering the specific fatty acids and their groups . In this (1)–(4) . Moreove (1)–(4) .(1)AI= values were estimated for each extract by applying the radical scavenging percentages of the plant extracts as a response variable.Data were analysed using the R platform  and RStuage in R , the conC. domestica and D. angustifolia had higher (p < 0.05) total phenolic content (TPC) compared to T. schimperi. The TPC obtained in the extract of C. domestica agreed with the ones reported from India for Curcuma longa [the methanolic extract of T. schimperi in the present study agreed with the report of [D. angustifolia was lower compared to the one previously reported [The phenolic content and antioxidant capacity of the methanolic plant extracts are reported in ma longa  but was ma longa . The obseport of  but was eport of . Howeverreported .T. schimperi and D. angustifolia had higher (p < 0.05) efficiency in scavenging DPPH radical with respective IC50 values of 33.97 and 25.59 µg/mL than the C. domestica extract (p < 0.05) . The IC50 values of D. angustifolia agreed with the previously reported ones [50 values of T. schimperi and D. angustifolia were comparable with ascorbic acid and Trolox reference antioxidants in scavenging DPPH and ABTS radicals, respectively (p > 0.05). Among the three plant extracts,  < 0.05) . Here, heport by . The D. ted ones . SimilarC. domestica had the lowest antioxidant capacity, while having a relatively higher phenolic content  Ascorbic Acid Equivalent (AAE) antioxidant capacity towards scavenging DPPH radicals compared to those in T. schimperi and D. angustifolia extracts with 221.81 and 236.53 µM AAE/mg of dry extracts, respectively  in diets supplemented with D. angustifolia, T. schimperi, and C. domestica extracts , compared to those with no antioxidant sources . Furthermore, the equivalent antioxidant power obtained between plant extracts and vitamin E supplemented diets (p > 0.05) in scavenging DPPH free radicals. However, the vitamin-E supplemented diet was superior (p < 0.05) in scavenging ABTS radicals compared to plant extract-containing diets  (The phenolic concentration and antioxidant capacity of the test diets are reported in and CD8) . Chickens face oxidative stress from various factors such as the inclusion of higher dietary fats . The supp > 0.05) effect on breast muscle fat content. Previously, a fat-lowering effect in breast muscle was reported in broilers fed with flaxseed and turmeric rhizome powder [p > 0.05) on breast muscle C14:0 and C16:0 contents. Among the extracts, TS8-fed hens had the lowest C4:0 and C18:0 content in breast muscle. The SFAs composition was reported to be less affected by dietary modification of the chicken diet compared to PUFA [The effects of feeding flaxseed, along with plant extracts and/or vitamin E on selected fatty acid contents in raw breast muscle of Sasso chickens, are presented in e powder . Among te powder , in this to PUFA ,73. In t to PUFA .p > 0.05) difference was observed in monosaturated fatty acids (MUFAs) contents between the tested diets. Previous reports were inconsistent regarding breast muscle MUFA content, in that [n−6 PUFAs, C18:2n − 6, and C20:4n − 6 contents were not affected (p > 0.05) in any of the diets. Moreover, the values are comparable to the previous report by [n − 6 PUFA and LA and an increase in C20:4n − 6 were reported in broiler chickens fed with 2 g linseed oil and 2 g sweet chestnut tannins [Moreover, no significant ( in that  found no in that  reported in that . However in that . In thiseport by  in broil tannins .C. domestica and T. schimperi significantly (p < 0.05) increased the level of ALA in breast muscle compared to the FS diet. Moreover, there was no significant difference observed (p > 0.05) in breast muscle ALA concentrations between hens fed with plant extract and vitamin E supplemented diets. Previous studies reported an increase [Amphora coffeaeformis in linseed oil-enriched diets, respectively. In contrast to the present study, feeding vitamin E along with 20% linseed oil increased the breast muscle ALA compared to diets supplemented with sweet chestnut tannins [Feeding hens with flaxseed along with extracts of increase  or no diincrease  in breas tannins .n − 3 (EPA) content was highest in CD8 (1.49 mg/100 g) and lowest in the NC diet (0.31 mg/100 g) (p < 0.05) the C22:6n − 3 (DHA) content in breast muscle in comparison to the NC diet (p > 0.05) was observed in breast muscle DPA (C22:5n − 3) content. However, the breast muscle DPA (C22:5n − 3) content was significantly increased (p < 0.05) in hens fed with TS8 and CD8, VE8 compared to NC and FS diets. Interestingly, the DPA concentration was the same between hens fed with plant extract incorporated feeds and vitamin E containing diet (p > 0.05) (n − 3 (DHA) in breast muscle compared to the NC diet (p < 0.05). Moreover, feeding chickens with CD8, but not the DA8 and TS8 diets, significantly (p < 0.05) increased the breast muscle DHA content compared to the FS diet. However, feeding chickens with DA8, TS8, and CD8 diets resulted in a significantly (p < 0.05) higher DHA content in the breast muscle compared to the NC diet (The breast muscle C20:5g/100 g) . Feeding NC diet . This li > 0.05) . Similar NC diet . The obsn − 3 PUFAs in breast meat [n − 3 (ALA) to LC n − 3 PUFA in chicken fed with plant extracts [Increasing the EPA and DHA contents is one of the most desired attributes of meat functionalization , these nast meat . Other sextracts  and planextracts ,36,75 inextracts  and sweeextracts  as an anp < 0.05) increased the n − 3 PUFA in breast muscle compared to the NC diet. The CD8 fed hens, but not the DA8 and TS8 diets had a significantly (p < 0.05) higher n − 3 PUFA content in breast muscle compared to the FS diet. Consistent with our findings, Kumar [n − 3 PUFA in the breast muscle of broilers fed with flaxseed along with turmeric rhizome powders. In contrast, no difference [n − 3 PUFA was reported in broilers fed with 2% grapeseed meal and 1.5–5% grapeseed oil in flaxseed enriched diets, respectively. The differences in breast n − 3 PUFA observed in this and across previous studies may be associated with plant species differences in active components. In the breast muscle, no difference (p > 0.05) in PUFA content was observed between treatment groups. Similarly, studies have shown no variation in breast PUFA in broiler chickens fed with flaxseed/linseed oil along with 0.5% of curry leaf, ginger or turmeric powders [Overall, feeding hens with FS significantly  in breast muscle EPA, DHA, and n − 3 PUFA contents between hens fed with vitamin E and plant extracts supplemented diets. However, feeding the hens with vitamin E elevated (p < 0.05) the breast muscle concentrations of EPA and DHA compared to those fed the FS diet. This might indicate the role of vitamin E and plant extracts as an antioxidant for preventing lipid peroxidation in feed and reducing oxidative stress in chickens. In agreement with our results, no difference in breast muscle EPA and DHA content was found between broiler chickens fed with 200 mg of vitamin E and 2 g sweet chestnut as natural antioxidants [The comparison of the current results showed no difference  content of heat-susceptible n − 3 LC-PUFAs, such as EPA (C20:5n − 3) and DHA (C22:6n − 3), were retained in cooked breast muscle in hens fed with FS compared to the NC diet (p < 0.05) EPA and DHA contents in cooked breast muscle compared to the FS diet. Henceforth, plant extract inclusion as an antioxidant might prevent the loss of oxidative susceptible and heat-sensitive EPA and DHA in breast muscle. Moreover, the EPA, DHA, and total n − 3 PUFA content in the cooked breast muscle from hens fed plant extracts were comparable to the ones fed with vitamin E (p > 0.05).In this study, a higher  n − 6/n − 3 ratio was observed in both raw and cooked breast muscle from chickens fed the FS diet compared to the NC diet  different between treatment groups. The breast muscle total MUFA, C16:1cis-9 and C18:1cis-9 contents were the highest in hens fed with DA8, but differences were not significant (p > 0.05) between the dietary groups. Similarly, the concentrations of LA (C18:2n − 6), ALA (C18:3n − 3), and DPA (C22:5n − 3) in cooked breast muscle did not differ significantly (p > 0.05) between the treatment groups. A recent comparative study in slow-growing leghorn chickens, reported a significant loss in breast muscle n − 3 PUFA during cooking, whereas the saturated fatty acids , as well as unsaturated fatty acids such as LA, ALA, and DPA contents, were less affected [p > 0.05) in cooked meat fatty acid profile between chickens fed with flaxseed as well as different antioxidants. In contrast, a respective decrease of 6.2% and 6.8% in SFA and MUFA in cooked chicken meat was reported in [In the cooked breast muscle, SFAs, C14:0, C16:0, and C18:0 contents were not significantly (affected  reportedorted in .p < 0.05) lowered the breast n − 6/n − 3 ratio compared to the FS diet . In a similar study, no difference was observed in breast muscle n − 6/n − 3 PUFA ratio between broilers fed with linseed oil and vitamin E and those fed with sweet chestnut tannins [n − 6/n − 3 ratio in the current finding was not lower than 4, which is the recommended ratio to prevent the occurrence of cardiovascular disorders [n − 6/n − 3 ratio may promote inflammatory, cancer, and cardiovascular risks [Feeding hens with TS8 in both raw and cooked and DA8 in raw breast significantly ( FS diet . A decree powder  and upone powder . In addi tannins . In geneisorders . Excess ar risks .p < 0.05) decreased the breast muscle thrombogenic indices (TI) compared to those fed with the NC diet (p > 0.05) between treatment groups with both raw and cooked breast meats. Similarly, the thrombogenic indices (TI) of breast meat (both raw and cooked) of chickens fed with plant extract containing diets and FS were not significantly different (p > 0.05). The AI, TI, and h/H ratio are health and nutritional value indicators of lipids [As per our findings, feeding hens with FS significantly , nutritional value indices (NVI), desirable fatty acids (DFA), and hypercholesterolemic fatty acids (HFA) in both raw and cooked breast muscle did not vary between dietary treatments. Comparable NVI, DFA, and HFA values were reported by  in similOxidative stabilityp < 0.05) in hens fed with plant extract antioxidant sources  compared to those fed with FS and the NC diets. A decrease in MDA concentrations by 52.04, 53.84, and 69.93% was observed in hens fed TS8, DA8 and CD diets, respectively, compared to the FS diet. Hens fed plant extracts had lower MDA values (1.53 to 1.71 µg/g) in raw breast muscle compared to fed control diets (2.00 to 2.60 µg/g). These lower MDA values could be attributed to the protective effects of plant polyphenol extracts against lipid peroxidation, as meat products with less than 3 mg MDA/kg sample are considered to be preserved from oxidative changes [n − 3 PUFA enriched cooked breast was reported by [p < 0.05) in both raw and cooked breast MDA was obtained in hens fed TS8 compared to the FS diet. Hence, an increase in MDA values in cooked breast muscle from hens fed diets with no antioxidant sources , indicates a lack of sufficient antioxidants to prevent lipid peroxidation. A rapid increase in lipid peroxidation has been observed in chicken meat heated at 70 °C and stored for 2 to 4 days [Oxidative deterioration in meat products is associated with the in vivo exposure of chicken to various stressors including diet. At the molecular level, excess oxidative stress lead to oxidative damage to important biological molecules including lipids . In this changes . In cookorted by . Overallo 4 days .p > 0.05). In agreement with the present finding, no difference was found in breast muscle MDA value between broilers fed with 2 g sweet chestnut tannins and 200 mg vitamin E as antioxidants in a linseed oil-enriched diet [Generally, the inclusion of plant extracts was found to reduce lipid peroxidation as observed in a previous study . Moreovehed diet .n − 3 PUFA in breast muscle is a direct consequence of plant extracts inclusion in the diet or it rather indirectly resulted from the reduction of oxidative stress. Feeding hens with plant extract in CD8 and VE8 significantly increased DHA and EPA contents, while DA8 and TS8 elevated EPA in raw breast muscle. While the SFA and MUFA contents were not influenced, a higher content of EPA and DHA was retained in cooked breast muscle . Moreover, feeding hens with diets without plant polyphenol extracts  increased the lipid peroxidation in raw breast muscle, whereas the inclusion of plant extracts decreased it. Feeding plant extracts in DA8, TS8, and CD8 diets showed superior efficiency in decreasing lipid peroxidation in breast muscle compared to feeding with vitamin E supplements. Further research is needed to verify whether the observed increase in


The synthesized LDH and LDH-TiO2 composite samples were characterized using the X-ray diffraction (XRD), field emission scanning electron microscopy (FESEM), and thermogravimetry analysis (TGA) methods. The XRD results for the uncalcined LDH indicated a hydrotalcite mass with a rhombohedral structure, whereas increasing the calcination temperature indicated transition to an amorphous state. FESEM results for the LDH-TiO2 matrix indicated round titanium dioxide particles and LDH hexagonal layers. The TGA findings for uncalcined LDH showed a gradual decrease in weight up to 250 °C, followed by a short plateau and then a sharp decrease in LDH weight from 320 °C, with a net weight loss around 47%. Based on the characterization and initial selenocyanate adsorption results, the 250 °C calcined LDH-TiO2 matrix was used for the selenocyanate photocatalysis. A ~100% selenium removal was observed using LDH:TiO2 at a 1.5:1 w/w ratio with a 2 g/L dose, whereas up to 80% selenium removal was noted for LDH:TiO2 at a 0.5:1 w/w ratio. The respective difference in the efficiency of selenium treatment was attributed to enhanced LDH-based adsorption sites in the enhanced LDH:TiO2 w/w ratio. Furthermore, the selenite and selenate that occurred during SeCN− photocatalytic degradation (PCD) were also nearly completely removed via adsorption. An optimization exercise using response surface methodology (RSM) for total selenium removal showed R2 values of more than 0.95, with a prediction accuracy of more than 90%. In summary, the present findings show that the use of a photocatalysis-cum-adsorption system based on LDH-TiO2 is a promising technique to treat industrial wastewater discharges for selenocyanate and also remove the resulting intermediates.Selenium as a nutrient has a narrow margin between safe and toxic limits. Hence, wastewater discharges from selenium-containing sources require appropriate treatment that considers health concerns and stringent selenium-related water treatment standards. This work examined the use of a photocatalysis-cum-adsorption system based on a layered double hydroxide coupled with TiO Some specific selenocyanate sources include mining facilities, large fossil fuel-based setups, and petroleum refineries [Ever-growing water pollution is a serious concern requiring appropriate environmental protection plans ,2. To thfineries ,5,6.2-initiated photocatalytic degradation (PCD) systems are also reported to be efficient for aquatic pollution control [2 matrix has also been demonstrated for aqueous pollution control. Seftel et al. (2010) [2 matrix as compared to that of TiO2 alone for the photocatalytic removal of methylene blue. Carja et al. (2010) [2/ZnLDH for treating aqueous phenol. Furthermore, Paredes et al. (2011) [2/LDH matrix produced a synergistic effect causing both higher ∙OH radical production and higher degradation of phenol than TiO2 alone. Similarly, the application of an LDH-TiO2 matrix for dimethyl phthalate and methylene blue pollutant removal has also been reported [2 matrix for the removal of 2,4-dichlorophenoxyacetic acid and orange II [2 system. Furthermore, though the use of LDH for selenite and selenate treatment is reported [2 matrix for the removal of selenocyanate (SeCN−) and associated selenite/selenate oxyanions. In addition, as mentioned earlier, aqueous phase selenocyanate (SeCN−) found in several industrial effluents is typically difficult to treat and requires specific treatment procedures. Hence, considering the recalcitrant nature of selenocyanate and the respective treatment challenges [2 matrix for selenocyanate removal along with the effect of different operational variables on process efficiency. This application offers a unique solution where the photocatalysis oxidizes selenocyanate to selenite and selenate followed by their uptake by the LDH-TiO2 matrix. Thus, the combined “photocatalysis-cum-adsorption” system offers a two-in-one solution. This study will also explore the possible reaction intermediates along with process optimization using response surface methodology (RSM)-based modeling.The use of layered double hydroxides (LDHs) has been reported for several environmental pollutant treatments, including arsenic , heavy m control ,30,31,32. (2010)  report h. (2010)  report t. (2011)  report treported ,36, as wrange II ,38. Thisreported ,40,41,42allenges , the preHigh-purity chemicals used were aluminum nitrate nonahydrate , titanium dioxide , magnesium nitrate hexahydrate , potassium selenocyanate , sodium selenite , and potassium selenate .2+:M3+) were transferred to 50 mL of distilled water and then stirred in an oil bath at 60 °C for about 15 min at 600 rpm, with pH adjusted to 10 ± 0.5 using NaOH (1 M). This was followed by stirring at 900 rpm with a temperature adjustment to 90 °C, and later on refluxing the suspension for 24 h and then aging it for 4 days at 80 °C [2 matrix was prepared as reported earlier [2 suspension at LDH:TiO2 ratios of 1.5:1, 1:1, and 0.5:1. This was first followed by mixing for 48 h at room temperature and then centrifuging and drying at 80 °C. The resulting LDH:TiO2 matrix was then calcined at 250 °C for 5 h and appropriately stored. The synthesized adsorbents were characterized using X-ray diffraction , field emission scanning electron microscopy , and thermogravimetry analysis . The XRD 2θ analyses were completed at a scanning rate of 3°/min from 5° to 70°. For the FESEM analyses, the respective materials to be analyzed were first coated with gold to make the surfaces conductive. The TGA analyses were completed in a nitrogen atmosphere from 50 to 800 °C at a step rate of 15 °C/min.The MgAl-LDH (hereupon referred to as LDH) was synthesized using a co-precipitation technique. Magnesium nitrate hexahydrate and aluminum nitrate nonahydrate at a molar ratio of 3:1 . The column used for the IC analyses was “Anion Dual 2” and the eluents used were 1.3 mM Na2CO3 and 2 mM NaHCO3.The layout of Pyrex glass reactor used for the PCD studies is given in As shown in the 2 matrix samples were characterized using several advanced techniques. The XRD profiles of uncalcined TiO2, uncalcined LDH, and calcined LDH-TiO2 (250 °C), are shown in 2 /2) and a is equal to 2d110). The basal spacing (d003) of 0.807 nm indicates nitrate in LDH as revealed by the presence of nitrogen in the EDX spectra analysis . Furthermore, it was also noted that using only TiO2 without UV had negligible effect on the SeCN− removal. Moreover, using UV with TiO2 led to the complete transformation of SeCN− into SeO42− within 6 h of UV irradiation, but no removal of SeO42− occurred. Hence based on this, along with the findings from the surface characterization exercise  of selenocyanate species. It is also important to note that immediately upon LDH-TiO2 sample addition before PCD, the pH increased approximately to 9 as also noted earlier [Initially, a preliminary set of adsorption experiments was completed to evaluate the selenocyanate adsorption capacity of synthesized LDH:TiOexercise , the 250 earlier ,61, whic earlier ,63,64,65 earlier :(1)Mg(1−+) species [− →SeO32−→SeO42−), while CN−, due to oxidation carried out by photogenerated holes (h+), converts to OCN−, as given below in Equations (3)—(5) [Furthermore, during photocatalysis, because of active species including ∙OH radicals and hole —(5) :(3)SeCN−− results  was also noted to fit to the classical Langmuir model (eqC (mg/L), eqQ (mg/g), mQ (mg/g), and b (L/mg) are the standard Langmuir isotherm parameters. The mQ for selenate was 14 mg/g, with similar values also reported earlier , indicating monolayer coverage for selenate adsorption onto LDH . The respective values of the slope, intercept, b, and adjusted R2 were found to be 0.07, 0.04, 1.8 L/mg, and 0.8946, respectively.ce sites . Neverth2 matrix can remove both the selenocyanate complex and the resulting selenium species during the combined photocatalytic treatment.The above findings show that the LDH-TiO42- residual, whereas for total selenium removal, the logit-transformed full quadratic model showed the best results. For the RS model (Equation (7)), the significance of the model terms with p-values < 0.05 shows that the respective terms significantly contribute toward improving the model results [2 has a p-value slightly higher than 0.05.The present work was expanded to further realize the effect of respective operational variables on selenium removal efficiency utilizing the response surface methodology (RSM)-based experimental design approach . To this results ; however2 values ;B=adsorbent dosage (1:2 g/L);C=selenocyanate concentration (2.5 to 7.5 mg/L);Residual SeO42−=residual concentration of selenate in solution after 6 h of UV irradiation (mg/L);TSR=selenocyanate removal efficiency expressed as total selenium removed (%).Both the RS (Equation (7)) and TS (Equation (8)) models show high R2 values , and theThe other statistical factors, including the normal plot of residuals C,D and t− 5 mg/L, enhanced LDH:TiO2 (L:T) initiates enhanced SeO42− removal with L:T 1.5 at 2 g/L delivering near-complete SeO42− removal (42− treatment efficiency can be attributed to enhanced LDH-based sites at an enhanced LDH:TiO2 (L:T) ratio. This is further corroborated by the fact that the variation in residual selenate and total selenium removal with the adsorbent dosage is reduced at the LDH:TiO2 1.5 ratio rather than at 0.5 .The PCD model optimization process generated 39 solutions for the stated objective function, with the desirability varying from 0.890 to 0.776, out of which the optimum removal for 7.5 mg/L SeCN2 0.5:1 displays a parabolic variation .The desirability function for LDH-TiOariation  owing to2 matrix for aqueous phase selenocyanate (SeCN−) treatment using photocatalytic degradation (PCD) and also extended the characterization results for synthesized LDH and LDH-TiO2 samples along with response surface methodology (RSM)-based modeling findings. The XRD results for the uncalcined LDH indicate a hydrotalcite mass with a rhombohedral structure, whereas the FESEM results for the LDH-TiO2 matrix indicate round titanium dioxide particles and LDH hexagonal layers. Based on the characterization and initial selenocyanate adsorption results, the 250 °C calcined LDH-TiO2 matrix was used for selenocyanate photocatalytic degradation. A ~100% selenium removal was observed using an LDH:TiO2 ratio of 1.5:1 with 2 g/L dose, whereas up to 80% selenium removal was noted for the LDH:TiO2 at a ratio of 0.5:1. Furthermore, the selenite and selenate that occurred during SeCN− photocatalytic degradation (PCD) were also almost completely removed via adsorption. In addition, both the residual selenate (RS) and total selenium removal (TS) RSM models yielded high R2 values, and the differences between the adjusted R2 and predicted R2 values were less than 0.2, which is also indicative of good prediction accuracy. This suggests that the respective RSM model predictions closely follow the experimental values.The present study employed the LDH-TiO


The total viable counts (TVC), psychrotrophic bacterial count, sensory index, texture analysis, and total volatile basic nitrogen (TVB-N) revealed that PL, MAP, and MAP + PL treatment delayed the deterioration of greater amberjack filets. These treatment groups also showed decreased accumulation of biogenic amines. High-throughput 16S rRNA gene sequencing results indicated that these treatments suppressed the growth of Pseudomonas in greater amberjack filets. Furthermore, the MAP + PL treatment group was observed to be more effective than the PL and MAP groups, extending the shelf life of greater amberjack filets by 6 days. This investigation showed that the combination of PL and MAP has the potential to retain the quality and extend the shelf life of greater amberjack.The combined effect of ε-polylysine (PL) and modified atmosphere packaging (MAP; 60% CO Seriola dumerili) is a cosmopolitan seawater fish species, and its farming and consumption have dramatically increased in recent years  during superchilling storage, whereas the microbiologically exceeded acceptable levels on day 26 in the air packing groups (2/40% N2) treatment groups was slower than in air packing groups during chilled storage, which could effectively maintain the quality of Atlantic salmon filets  is an effective strategy for food preservation that inhibits microbial growth, decreases lipid oxidation, and protein degradation , 6. Prev with O2 , 8–10. Tn filets . ε-polyl albulus . PL has s aureus , 14, whis aureus . The abiith O2 2/0% N2 durs aureus , 17. HowTo address this deficiency, the effects of PL, MAP, and their combination on the physicochemical indicators of farmed greater amberjack filets during storage at 4°C were evaluated in this study. Changes in the microbial community were investigated by high-throughput 16S rRNA gene sequencing. Understanding the effects of PL, MAP, and combination treatment on the changes in microbiological and physicochemical properties is aimed at providing an effective preservation strategy for extending the shelf life of farmed greater amberjack filets.2 and 60% N2 gas mixture were injected, and the bags were sealed using a packaging machine . The initial gas and filet ratio were approximately 4:1 (v/w) for the atmospheric air and MAP treatment groups to ensure a relatively stable composition of gas during greater amberjack storage. Specifically, control and PL groups were packaged in a normal polypropylene bag, while MAP and MAP + PL groups were packaged in high gas barrier performance bags with a low oxygen transmission rate of 0.42 cm3 m–2 day–1 atm–1 at 22°C . For each treatment group, a total of 120 individually packed filets were prepared and stored at 4°C for 12 days. Sampling bags were randomly selected from each group at each sampling time.Farmed greater amberjack (weighing 2200 ± 120 g) was purchased from a local aquatic market in Guangzhou, China, and transported to our laboratory on ice in a large tank within 30 min. The greater amberjack was slaughtered using head shot, scaled, gutted, deskinned, and sliced into filets on a clean bench (weight of 200 ± 10 g). Moreover, all the processing steps were carried out with sterilized tools, and all workers wore sterilized masks and gloves. The filets were then washed in sterilized water, drained through sterilized stainless steel wire mesh for 5 min, and randomly separated into four groups. The four groups were performed as follows: (1) control; filets were soaked in sterilized water for 15 min, drained on a clean bench for 10 min on sterilized stainless steel wire mesh, and packaged with atmospheric air. (2) PL; filets were soaked in 0.5% (w/v) PL for 15 min, drained, and packaged with atmospheric air. (3) MAP; filets were soaked in sterilized water for 15 min, drained, and packaged with mixed gas. (4) MAP + PL; filets were soaked in 0.5% (w/v) PL for 15 min, drained, and packaged with mixed gas. Sterilized soaking water and PL solutions were prepared at 4°C, and the filet/soaked solution ratio was 1:5 (w/v). Food grade PL (purity ≥ 99%) was purchased from Bainafo Biotechnology Co., Ltd. . The bags in the control and PL groups were sealed and packaged with atmospheric air. Air was first removed from the MAP and MAP + PL groups, then the 40% CO® 3500, Thermo Bioreactor, Seward Ltd., Worthing, UK). The homogenized solutions were serially diluted with 0.01 M PBS (pH 7.2) and 100 μL of the resultant serial solution was spread onto Tryptic soy agar plates . The plates were incubated at 25°C for 48 h for total viable counts (TVC), and at 7°C for 10 days for psychrotrophic bacterial counts, respectively.The microbial analysis was performed using the method described by Li et al. . At eachg for 5 min at 4°C. The pH of the supernatant was measured using a digital pH meter . Moreover, 5 mL of the supernatant was equally mixed with 10 g/L of magnesium oxide in a Kjeldahl tube, and total volatile basic nitrogen (TVB-N) was measured using the Kjeldahl nitrogen apparatus , as previously described (The greater amberjack samples (10 g) were minced using a hand blender . Five grams of minced meat was homogenized with 50 mL of deionized water for 30 s using a vortex mixer  and shaken on a gyratory shaker at 100 rpm for 30 min at room temperature. The homogenized mixture was centrifuged at 2,800 × escribed . The resThe sensory analysis of the fish filet was performed according to a previous study . Ten trag for 10 min. The supernatant (1 mL) was mixed with 100 μL of 2 M sodium hydroxide and 300 μL of saturated sodium bicarbonate, and 1 mL of dansyl chloride was added (10 mg/mL), followed by incubation at 40°C for 45 min in the dark for derivatization. After the derivative reaction, biogenic amines were analyzed using HPLC. The 20 μL of samples solutions were injected into HPLC  equipped with a reversed-phase chromatographic column  and an SPD-M20A ultraviolet detector  at 254 nm wavelength. The 0.1 M ammonium acetate and 100% acetonitrile were used as gradient elution solvent A and solvent B. Gradient elution program was performed as previously described  and a TA44 probe (4-mm diameter) was used to measure the fish sample pieces (2 cm × 2 cm × 1 cm). Hardness, chewiness, and springiness were measured by texture profile analysis (TPA) mode with test speed of 1 mm/s, test distance of 5 mm, and trigger force of 5.0 g . Each sag for 7 min to remove fish particles, and the supernatant was obtained and centrifuged at 12,000 × g for 10 min at 4°C. The resulting pellet was used for DNA extraction. DNA was extracted using the Magnetic Soil and Stool DNA Kit , according to the manufacturer’s protocol. DNA quality was checked using 1% agarose gel electrophoresis. The 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-ACCTTGTTACGACTT-3′) primers were used to amplify the 16S rRNA gene using Kapa HiFi HotStart ReadyMix . Each 20 μL of PCR mixture contained 10 ng template DNA, 2 μL of dNTPs (2.5 mM), 0.4 μL of DNA polymerase (5 U/μL), 4 μL of 5 × PCR buffer, 0.8 μL of forward primer (5 μM), and 0.8 μL of reverse primer (5 μM). The PCR amplification conditions were as follows: 95°C for 2 min, 30 cycles of 95°C for 30 s, 55°C for 30 s, 72°C for 1 min 30 s, and a final extension for 7 min at 72°C. The PCR amplicons were purified using Agencourt AMPure XP Beads  and quantified using a Qubit 4.0 fluorometer . Subsequently, the SMRT bell libraries were constructed from PCR amplicons using the SMRT bell Template Prep Kit 2.0, according to the manufacturer’s instructions (Pacific Biosciences). Purified SMRTbell libraries from the pooled and barcoded samples were sequenced using the Sequel II Sequencing kit 2.0 on a single PacBio Sequel II 8M cell. The raw reads were filtered and demultiplexed using SMRT Link software (version 8.0) (PacBio) to obtain circular consensus sequencing (CCS) reads. CCS reads were sorted into different samples based on barcodes. After removing the primer and barcodes, high-quality sequences were extracted using the Quantitative Insights into Microbial Ecology (QIIME) package (version 1.7) (1 Taxonomy of the OTUs was classified using Greengenes databases (version 13.8)   and UCLUion 1.7)  were useion 1.7) . Potention 13.8) , Ribosomon 13.8) , and SILon 13.8)  with a mP < 0.05.All measurements were performed at least in triplicate and are presented as mean ± standard deviation. The data were analyzed using one-way ANOVA with multiple comparisons of Tukey HSD tests. Statistical significance was set at 2/30% N2 extended the microbiological shelf life of golden pompano filets by 5 days (Aristichthys nobilis) (Litopenaeus vannamei) (Pseudosciaena crocea)  , Pacificannamei) , and larm growth –38. All The changes in physicochemical properties and sensory parameters, including pH, TVB-N, and SI for the control, PL, MAP, and MAP + PL groups are shown in The results of the sensory analysis are shown in 2 conditions, leading to low TVB-N values. The TVB-N value in the MAP + PL groups did not exceed the acceptable levels after 12 days of storage, but the SI was lower than the set acceptable level of 2.5. This result corresponds with the previous studies, which also reported that TVB-N was low despite sensory analysis results indicating fish spoilage  has become the dominant bacterial at 6 days of storage. Pseudomonas was relatively rare in the control group before storage but gradually increased with storage time to 40.3% becoming the predominant species in the control group at the end of storage at 12 days. Pseudomonas (19.95%), Serratia (29.60%), and Acinetobacter (23.36%) became dominant in the PL treatment group at the end of storage. Acinetobacter is an aerobic species, which was usually found in flesh fish or in the middle or end of storage but not as the dominant spoilage bacterial  can be found below: DW: data curation, methodology, validation, writing – original draft, visualization, and funding acquisition. XL: data curation, methodology, and formal analysis. XY: project administration and supervision. SC and YW: methodology and software. LL: formal analysis and writing – review and editing. CP: formal analysis and resources. YZ: writing – review and editing and funding acquisition. All authors contributed to the article and approved the submitted version.


Metastatic breast cancer poses great challenge in cancer treatment. N‐dihydrogalactochitosan (GC) is a novel immunoadjuvant that stimulates systemic immune responses when administered intratumourally following local tumour ablation. A combination of photothermal therapy (PTT) and GC, referred to as localized ablative immunotherapy (LAIT), extended animal survival and generates an activated B cell phenotype in MMTV‐PyMT mouse mammary tumour microenvironment (TME). However, how T cell populations respond to LAIT remains to be elucidated.+ and CD4+ T cells to the therapeutic effect of LAIT. Using single‐cell RNA‐sequencing (scRNAseq), we analysed tumour‐infiltrating T cell heterogeneity and dissected their transcriptomes upon treatments of PTT, GC, and LAIT (PTT+GC).Using depletion antibodies, we studied the contributions of CD8+ T cells after LAIT abrogated the therapeutic benefits of LAIT. Ten days after treatment, proportions of CD8+ and CD4+ T cells in untreated TME were 19.2% and 23.0%, respectively. Upon LAIT, both proportions were increased to 25.5% and 36.2%, respectively. In particular, LAIT increased the proportions of naïve and memory cells from a resting state to an activated state. LAIT consistently induced the expression of co‐stimulatory molecules, type I IFN responsive genes, and a series of antitumor cytokines, Ifng, Tnf, Il1, and Il17 in CD8+ and CD4+ T cells. LAIT also induced immune checkpoints Pdcd1, Ctla4, and Lag3 expression, consistent with T cell activation. Relevant to clinical translation, LAIT also upregulated genes in CD8+ and CD4+ T cells that positively correlated with extended survival of breast cancer patients.Loss of CD8Overall, our results reveal that LAIT prompts immunological remodelling of T cells by inducing broad proinflammatory responses and inhibiting suppressive signalling to drive antitumour immunity. LAIT induced a broad proinflammatory response in tumour‐infiltrating T cells. LAIT causes tumour immunogenic cell death (ICD), releasing danger associated molecular patterns (DAMPs) and tumour antigens (TAs). DAMPs and TAs recruit T cells into the tumour microenvironment, and the GC‐induced cytokines drive their differentiation into T helper 1 and 17 (Th1 and Th17) cells and cytotoxic lymphocytes (CTL). Localized ablative immunotherapy (LAIT) was developed to achieve this objective. LAIT has two components: local photothermal therapy (PTT) and local administration of an immunostimulant, such as N‐dihydrogalactochitosan (GC).To elucidate the factors contributing to T cell activation following LAIT, we used single‐cell RNA sequencing (scRNAseq) to evaluate the immunological effects of LAIT on breast tumours arising from the mouse mammary tumour virus‐polyoma middle T (MMTV‐PyMT) transgenic mice. MMTV‐PyMT tumours have high penetrance and emulate the histological stages of human luminal type B breast cancer.+ and CD8+ T cells in the TME in response to treatment. Using single cell trajectory analysis, we uncovered that GC and LAIT treatment pushed the tumour resident T cells into an activated state from a resting state. Complementing these findings gene expression analysis also revealed that LAIT consistently induced genes that enriched pathways involved in co‐stimulation, responses to type I IFN, and antitumor cytokine production including Ifng, Tnf, Il1, and Il17 across the T cell subtypes. In support of the activated T cell phenotype, we also observed induction of immune checkpoints of Pdcd1 and Ctla4, indicating that LAIT has great potential to synergize with ICI. More clinically relevant, LAIT upregulated genes in CD8+ T and CD4+ T cells that positively correlated with extended survival of breast cancer patients. Overall, our results reveal that LAIT prompts immunological remodelling of T cells by inducing broad proinflammatory responses and inhibiting suppressive signalling to drive antitumor immunity. This study provides the rationale for synergizing LAIT with ICI to overcome the inhibitory mechanisms imposed by the TME, therefore increasing the therapeutic efficacy of both treatments.In this study, we sought to determine if LAIT disruption of the established TME could alter the transcriptome of tumour‐infiltrating T cells and allow for immune mediated tumour killing. T cells from untreated (CTRL), PTT, GC, and LAIT (PTT+GC) treated MMTV‐PyMT tumours were evaluated. Single cell transcriptomes revealed a dynamic atlas of CD422.13, mice were divided into four treatment groups: Untreated controls (CTRL), PTT alone, GC alone, and LAIT (PTT+GC) (n = 3–4 per group). Since cytotoxic T cells are crucial to antitumor immune responses, we evaluated the extent of cytotoxic T cell induction by LAIT in the TME 10 days after treatment by flow cytometry. Thermal effects of laser irradiation reduce tumour size, resulting in significantly smaller tumours in the PTT and LAIT groups than that in CTRL and GC groups  and memory T cells (CD45+CD3+CD8+CD44HighCD62L+) in LAIT and GC groups had a trending increase, while the CD45+CD3+CD8+CD44LowCD62L+ and CD45+CD3+CD8+CD62L+ naïve T cells in LAIT and GC groups were like that of CTRL and PTT groups  promoter‐driven PyMT (polyoma virus middle T) tumour antigen develop breast cancer which closely resembles human pathogenesis.s Figure . For com3 Figure , followis Figure .+ T cell populations, using the gating strategy in Figure + T cells from LAIT‐treated tumours based on their distribution and number of distinct clusters . The effector cells  from the LAIT‐treated tumours also separated into distinct clusters, with a much greater distance between them on the t‐SNE plot  and an increase in the CD28+CD69+ cells (red dots) in the LAIT‐treated tumours, suggesting the differentiation and activation of CD8+ T cells following LAIT  T cell subsets, were statistically elevated in the LAIT‐treated tumours compared to GC alone and trending up compared to CTRL and PTT  are critical for downstream responses following LAIT  cells play a significant role in tumour progression and metastases, as loss of CD4+ T cells prohibited lung metastases in untreated MMTV‐PyMT tumour bearing animals.+ T cells are imperative to mounting an anti‐tumour response in this model. This should not diminish the important role of CD4+ T cells in generating T cell immunity but indicates that the timing of CD4+ T cell depletion likely changed the dependence on CD4+ T cells in this model. Moreover, this is a virally driven oncogene model, and as such a strong CD8+ T cell response likely exists well before the depletion of CD4+ T cells as these tumours were implanted first and allowed to reach a specific size before T cell depletion. Thus, the help of CD4+ T cells is not as critical for acute stimulation of a memory response, as corroborated by individual tumour growth in which the CD4+ T cell depleted animals had significant tumour regression initially . CD45+ cells from two separate animals were pooled together in equal numbers and then used for sequencing. First, all 49 380 TILs  were integrated for scRNAseq by Seurat R package.To investigate a broad spectrum of immune cell compositions and transcriptional profiling in the TME 10 days after treatment, scRNAseq analyses were performed following the workflow depicted in Figure + T cells (cluster 0) and central‐memory CD4+ T cells (cluster 9), as shown in Figure + T cells , NK cells (cluster 4), the combined naïve CD4+ T cells (cluster 5 and 6), CD4+ Tregs (cluster 11), and central memory CD4+ T cells (cluster 9). Furthermore, mTORC1, G2M checkpoint, and Myc target pathways were downregulated by LAIT in these cell clusters . Using differentially expressed genes (DEGs), we focused on the comparison between the LAIT and CTRL groups. As shown in Figure s Figure . The sig+ T cells from clusters 0 and 3 with CD4+ T cells from clusters 5, 6, and 9, and γδ T cells in cluster 1. In comparison, 65, 16, and 5 commonly shared DEGs were found in LAIT versus CTRL, LAIT versus PTT, and LAIT versus GC, respectively , Isg15, and Ptprc, which are involved in T cell survival, activation, and death  analysis, using R package + Figure  and CD4+s Figure . The CD8s Figure  with the6 Figure . Non‐naï9 Figure . When ses Figure . The gena Figure . These gs Figure . Overalls Figure .+ T cells  were generated by using FindMarkers function in Seurat R package.p value <.05, shown as red and blue dots in the volcano plots  were enriched in exhausted, cycling exhausted, and effector memory CD8+T clusters, with LAIT only slightly elevating levels of Ifng and Tnfsf10. Co‐inhibitor molecules of Pdcd1 (PD1), Ctla4 (CTLA4), and Lag3 (LAG3) were mainly expressed in exhausted, cycling exhausted and effector memory CD8+ T clusters with Pdcd1 showing slight induction by LAIT  was ubiquitously expressed and sharply upregulated by LAIT in naïve and memory CD8+ T cells. Downstream molecules, including transcription factors Stat1, Stat4, and Stat6, were activated by treatments of PTT, GC, and LAIT with variable degrees. Irf3 and Irf7 were strongly induced by LAIT in activated and naïve memory CD8+ T cells, respectively. Interferon induced genes, including Ifi47, Ifi206, Ifi208, Ifi209, and Ifit3, were markedly stimulated by LAIT in naïve memory CD8+ T cells. Interferon stimulated genes Isg15 showed high promotion by LAIT in naïve memory, activated and cycling CD8+ T cells. In exhausted and effector memory T cells, Ccl4 and Il17a were upregulated by treatments of PTT, GC, and LAIT, and proinflammatory genes Il1b and S100a9 were also strongly induced by LAIT. These results illustrate that LAIT selectively stimulates T cell activation and IFN related pathway genes in CD8+ T cell subpopulations  were enriched in non‐naïve CD4+ T cell types, with Ifng being strongly driven by GC and LAIT. In activated CD4+ T cells, Ifng and Tnf was strongly induced by GC and LAIT suggesting that these treatments drive a T helper 1 response in activated T cells , Ctla4 (CTLA4), Lag3 (LAG3), Havcr2 (TIM3), and Entpd1 (CD39) were mainly expressed in non‐cycling and cycling Treg clusters with Lag3, Havcr2, and Entpd1, showing slight induction by GC and LAIT. Treg genes Foxp3 and Il2ra (CD25) also showed a similar regulation pattern. Type I IFN response in Tregs has been shown to mitigate Treg functionality.Gzmb, cytotoxic cytokines, and ICIs suggesting that the tumour Tregs were also highly activated by our treatment and thus work against immune mediated killing of the tumour cells. This hypothesis was corroborated by the fact that CD4 depletion prior to LAIT treatment enhances tumour killing for a period  and Ifih1 (MDA5) were ubiquitously expressed and stimulated by all treatments with sharp upregulation by LAIT in all subtypes of CD4+ T cells. Their positive regulator Ddx60, downstream molecules, including transcription factors of Stat1, Stat2, Stat6, Irf1, Irf2, Irf3 and Irf7, were activated by treatments of PTT, GC, and LAIT with variable degrees. Interferon stimulated and induced genes, including Isg15, Isg20, Ifi47, Ifi203, Ifi206, Ifi208, Ifit3, and Ifi27l2a, showed similar regulation pattern by treatments and were markedly stimulated by GC and LAIT in all subpopulations of CD4+ T cells , in activated CD4+ T cells, Tregs and cycling Tregs. Tgfbr2 was downregulated by LAIT in all subtypes of CD4+ T cells. Inhibition of Foxo1, Foxo3, Hif1a, Stat3, Stat5a by treatments of PTT, GC, or LAIT were seen in almost all subsets of CD4+ T cells, except that Stat5a showed a slight increase in cycling Tregs. Another panel of heat shock proteins, including Hspa1a, Hspe1, Hspa8, Hspa4, Hspd1, and Dnajb6, were also downregulated by treatments, especially by LAIT, across all the CD4+ T subpopulations  based on gene set variation analysis (GSVA).+ and CD4+ T cells exhibited prolonged overall survival compared to the patients with lower ES score of such gene set expression  in exhausted and effector memory CD8+ and CD4+ T cells. The induction of immune checkpoints of Pdcd1 and Ctla4 by LAIT ,Clinically, the key to successful tumour treatment depends on the effective reversal of the immunosuppressive TME. Low numbers of tumour‐infiltrating T cells and high numbers of immunosuppressing cells or immature myeloid cells leads to a poor prognosis, treatment resistance, and low overall survival rates of cancer patients.Our previous work demonstrated that LAIT can globally alter the TME and establish a proinflammatory phenotype in tumour‐infiltrating B cells.44.1Female FVB/NJ wild‐type (stock #001800) and FVB/N transgenic MMTV‐PyMT (stock #002374) mice were purchased from Jackson Laboratories and housed according to institutional guidelines at the University of Oklahoma Health Sciences Center according to the descriptions in our previous work.4.2MMTV‐PyMT murine breast tumour organoids were isolated from FVB/N‐Tg (MMTV‐PyVT) 634Mul/J mice as previously described.4.33, the tumour site was shaved and mice received one of four treatments: CTRL (without treatment), GC alone, PTT alone, and LAIT (PTT+GC). The mice in GC group received an intratumoural injection of 1% GC in 0.1  ml solution . For mice in PTT group, the laser parameters were selected based on our previous studies.2 for 10 min, using an optical fibre with a diffusion lens  to deliver uniform light distribution on the treatment surface. For the mice in LAIT group, the tumour was treated by the laser (1 W/cm2 for 10 min), followed by an intratumoural injection of GC (0.1 ml at 1%). Ten days after treatment, tumours were collected from selected mice in each group. The remaining mice were observed for up to 60 days or when the tumours reached a size of 2.5 cm3 or reached ethical endpoints.Tumour‐bearing mice were treated according to the methods previously described.4.43, 400 μg of anti‐CD4, ‐CD8, or isotype antibody (BioXcell) was injected i.p. Twenty‐four hours after antibody injection, tumours were treated with LAIT or left untreated. Three days after LAIT treatment, mice were injected with 200 μg antibody, i.p. and maintained on this dose biweekly until tumours reached a size of 2.5 cm3 or reached ethical endpoints.Once tumours reached 0.4 cm4.5Tumour tissues were isolated, minced, and digested with Collagenase IV and DNase I at 37°C for 20–30 min in RPMI. After enzymatic digestion, immune cells were enriched using lymphocyte separation medium (Corning). The enriched cells were then used for flow cytometry analysis.4.6+ cells using the EasySep Mouse Streptavidin RapidSpheres Isolation Kit  and then sorted using the MoFlo sorting system based on the expression of CD45  and viability .All samples were run on the LSR II and data were analysed using FlowJo v10.6.1 . Briefly cells were stained on ice for 15–20 min with the viability dye Ghost Dye BV510  and the following antibodies: CD45 , CD8 , CD44 , CD4 , CD28 , CD62L , CD3 , CD69 , CCR7 , CD25 , Foxp3 , and CD103 . Cells were subsequently washed and fixed using the FoxP3 Transcription Factor staining buffer set . The cells used for scRNAseq were depleted of EpCAM4.7+ tumour‐infiltrating immune cells were encapsulated into droplets via a 10× Genomics platform according to the manufacturer's instructions. Paired‐end RNA‐seq was performed via an Illumina NovaSeq 6000 sequencing system. The Linux command cellranger count from Cell Ranger pipeline was used to process sample‐specific FASTQ files into feature‐barcode matrices. Seurat (v3.2) was used to process and integrate above scRNAseq datasets from different treatment groups.FindVariableFeatures function by setting feature numbers as 2000. Principal component analysis (PCA) was performed using the first 30 principal components (PCs). Clustering was performed using FindClusters that implements a shared nearest neighbour (SNN) modularity optimization‐based clustering algorithm with resolution 0.5 for default analysis. Lymphoid compartment cells were subset and re‐clustered with resolution 0.7 to generate its UMAP plot. The accession number for the scRNAseq data reported in this paper is GEO: GSE150675.Tumour sample processing, tumour‐infiltrating immune cell preparation, and single‐cell RNA sequencing barcoding and library generation were performed according to our previous work.4.8FindMarkers function in Seurat R package was used to obtain differential gene expression for each comparison with threshold of Log fold change (logFC) set at 0.25 and −0.25 as default.ggplot2 in R. clusterProfiler R package was adopted for gene functional enrichment analysis by using both over‐representation analysis (ORA) and gene set enrichment analysis (GSEA) methods and gene annotation and pathway databases including GO, KEGG, Reactome, and MsigDB.Treatments of PTT, GC, and LAIT (PTT+GC) were compared with CTRL, respectively. 4.9VennDiagram R package following with subset of indicated gene sets functional enrichment analysis.AverageExpression function in Seurat R packageTreatment‐derived DEGs were overlapped and visualized by using 4.10https://xenabrowser.net/datapages/) and used for assessing the clinical significance of expression of a given gene set.biomaRt R package.Gene expression RNAseq and survival data available from GDC TCGA Breast Cancer (BRCA) (20 datasets) were downloaded from UCSC Xena .Statistical analysis was performed with GraphPad Prism and R. Unless indicated otherwise, data were expressed as means ±SD or SEM. One‐way analysis of variance (ANOVA) was used for multiple group comparisons. Proportion test function (“prop.test”) in R was used for cell proportion test. Differences in survival were determined on the basis of Kaplan‐Meier survival analysis. Adjusted Wei R. Chen is co‐founder and an unpaid member of the Board of Directors of Immunophotonics, Inc. Tomas Hode and Samuel S. K. Lam declare a conflict of interest as employees with minority ownership stakes of Immunophotonics, Inc., the manufacturer of the proprietary immune stimulant GC. Other authors confirm that there is no known conflict of interest associated with this publication. There was no financial support that influenced the conclusions of this manuscript.https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE150675). Analysis of such data can also be available upon request.The accession number for the scRNAseq data reported in this paper is GEO: GSE150675 (Supporting InformationClick here for additional data file.


Bacillus cereus ADO11, Stenotrophomonas maltophilia NAA11, Acinetobacter pittii LAM11, and Serratia marcescens NSA15, which were isolated from maize soils and have the ability to stimulate plant growth. The genome assembly sizes for the strains were 4,476,462 bp, 4,731,402 bp, 4,080,875 bp and 4,959,744 bp, respectively.This study presents the draft genome sequences of four strains of rhizobacteria, namely,  Bacillus cereus ADO11, Stenotrophomonas maltophilia NAA11, and Acinetobacter pittii LAM11 strains were obtained from rhizospheric maize soils in the southern Benin agroecological zone between 6°30′N and 6°45′N and between 1°35′E and 2°45′E. The Serratia marcescens NSA15 strain was obtained from rhizospheric maize soils in the north Benin agroecological zone. To obtain samples, maize plant roots were cut, with the adhering soil, and mixed in a bucket. The soils of the maize rhizosphere were carefully placed in sterile bags in a cooler containing ice accumulators, transported, and stored at 4°C for 72 h in the Laboratoire de Biologie et de Typage Moléculaire en Microbiologie of the Université d'Abomey-Calavi, Benin for analyses. The soil samples were serially diluted by decimal dilution according to the method described by Speck  .JAIRCN000000000 (ADO11), JAIUDP000000000 (NAA11), JAMQVB000000000 (LAM11), and JAMQIW000000000 (NSA15). The versions described in this paper are the first versions, JAIRCN000000000.1 (ADO11), JAIUDP000000000.1 (NAA11), JAMQVB000000000.1 (LAM11), and JAMQIW000000000.1 (NSA15).This whole-genome sequencing shotgun project and associated data have been deposited in DDBJ/ENA/GenBank under the accession numbers listed in


Staphylococcus aureus (MRSA) is a multidrug-resistant pathogen that currently poses a serious threat to global health. Novel antimicrobial agents against MRSA are urgently being developed. In this study, we investigated WYBQ-4, which is an effective antibacterial agent with potent bactericidal activity and bactericidal efficiency against MRSA USA300 and clinical isolate strains. In addition, WYBQ-4 exhibited low cytotoxicity without hemolytic activity according to a safety evaluation. Importantly, WYBQ-4 showed potent in vivo efficacy in an MRSA-induced mouse pneumonia model, systemic infection model, and intramuscular infection model. The efficacy of this new cephalosporin against MRSA was associated with a high affinity for penicillin-binding proteins  evaluated in a competition assay using bocillin as a reporter. In conclusion, WYBQ-4 has a significant bactericidal effect in vitro and in vivo, indicating that it is a promising compound to control MRSA infection.Methicillin-resistant IMPORTANCE Antibiotic resistance is spreading faster than the introduction of new compounds into clinical practice, causing a public health crisis. Novel antimicrobial agents against MRSA are urgently being developed. In this study, we investigated WYBQ-4, which is an effective antibacterial agent with potent bacteriostatic activity and bactericidal efficiency against MRSA USA300 and clinical isolate strains. WYBQ-4 showed potent in vivo efficacy in MRSA-induced mouse models. Subsequently, we further revealed its antibacterial mechanism. In conclusion, WYBQ-4 has a significant bactericidal effect in vitro and in vivo, indicating that it is a promising compound to control MRSA infection. Staphylococcus aureus (MRSA) pose a significant public health risk to humans. MRSA is a major source of infection in hospitals and clinics, ranging from soft tissue infections to bacteremia, endocarditis, pneumonia, and osteomyelitis, and is a leading cause of morbidity and mortality worldwide  and inhibit S. aureus cell wall synthesis by inhibiting transpeptide action, which is similar to ceftobiprole  29213 , MRSA USA300 , methicillin-resistant Staphylococcus epidermidis (MRSE) 12228 , and methicillin-susceptible Staphylococcus epidermidis (MSSE) 35984 . In addition to Staphylococcus, WYBQ-4 was also found to be active against Streptococcus pyogenes , but the antibacterial activity against Enterococcus faecalis, Enterococcus faecium, and Gram-negative bacteria was weak compared to that against Staphylococcus.The spectrum of activity of WYBQ-4 was determined by measuring its MIC against a panel of Gram-positive bacteria and Gram-negative bacteria. As shown in in vitro  against clinically isolated MRSA and MSSA, respectively , 11. Amoin vitro  to G andin vitro  and H.S. aureus reached 109 CFU/mL after culturing for 9 h from an initial period of 106 CFU/mL and reached the stationary phase. According to the curves of the WYBQ-4-treated groups, obvious inhibition of bacterial growth was observed. When the concentration of WYBQ-4 reached 4× MIC, it showed complete sterilization on MSSA 29213 at 9 h . Similar results were also observed in bacterial asymmetric division and distortion of septa. In conclusion, these results indicated that WYBQ-4 causes cell wall thinning, septum distortion, and division asymmetry in S. aureus.Transmission electron microscope (TEM) assays were performed to further investigate the effect of WYBQ-4 on the morphology of S. aureus USA300 were determined using a competition fluorescence gel assay, and bocillin was used as the fluorescent ligand. As shown in To explore the antibacterial mechanism, the binding affinities of WYBQ-4 for PBP1, PBP2, PBP3, PBP4, and PBP2a of Since there is a direct interaction between WYBQ-4 and PBP2a, we simulated the key site and binding mode of WYBQ-4 binding to PBP2a by molecular docking. As illustrated in 0k, was firstly derived from the linear phase of absorbance variation with time, and then the apparent primary rate constant of the acylation reaction of nitrofuran at 100 μM was determined by absorbance variation with time. This procedure was calculated using the WolframAlpha website . The calculated secondary rate constant, 0k, for the nonspecific hydrolysis of nitrocefin is 2.3 ± 0.14 M−1s−1; the primary reaction rate constant, kn*, for 100 μM nitrocefin is approximately 0.05 s−1.The kinetics of WYBQ-4’s interactions with PBP2a have been evaluated. The nonspecific secondary rate constant, k2) is large but deacylation (k3) is very small  with 2k/sK of 174 ± 8.45 M−1s−1. In addition, the second-order rate constant for the PBP2a acylation by ceftaroline has been reported to be 4,500 M−1s−1. It is possible that ceftaroline’s k2/sK ratio was greater because conformational changes enabled it to open the active site of PBP2a, resulting in a more favorable acylation reaction. Subsequently, PBP2a (15 μM) and WYBQ-4 (0 to 90 μM) were included in the 60-μL reaction system for 45 min, and the free protein was detected by adding BOCILLIN FL when the reaction reached equilibrium, and the sK was found to be 28 ± 1.24 μM.Subsequently, nitrocefin (100 μM) was used as a reporter molecule to determine the apparent first-order rate constants for WYBQ-4 acylation at different concentrations (0 to 500 μM) in competition assays. Since the modified WYBQ-4 is based on the structure of cephamycin C, it should have an effect similar to that of other cephalosporin antibiotics, for which the acylation rate constant  and then treated with WYBQ-4 (75 and 37.5 mg/kg) (S. aureus pneumonia with no significant difference (P > 0.05).To evaluate the therapeutic activity of WYBQ-4 in the lung model, mice were challenged with USA300 (2 × 105 mg/kg) . Untreat5 mg/kg) . For the10 CFU/g in the infected mice without treatment. After treatment with 75 mg/kg WYBQ-4, the bacterial counts in the lung were reduced significantly to 5.44 ± 0.15 log10 CFU/g compared with untreated mice (P > 0.05). Thus, 75 mg/kg WYBQ-4 significantly reduced the number of surviving MRSA USA300 organisms in the lungs of mice.The lung tissue bacterial load of WYBQ-4-treated mice was also further assessed as shown in ted mice . There wTo investigate whether WYBQ-4 can ameliorate the lung damage caused by nonlethal exposure to USA300, the degree of damage to the lungs for different doses of WYBQ-4 was observed. Untreated infected mice showed some degree of acute injury characterized by interstitial hyperemia and edema with marked focal hemorrhage. After treatment with WYBQ-4 or ceftaroline fosamil, inflammatory signs of lung tissue were significantly reduced, with little infiltration of inflammatory cells. Moreover, WYBQ-4 (75 mg/kg) showed better efficacy than ceftaroline fosamil (75 mg/kg) in the treatment of infected lesions .S. aureus-induced pneumonia.Taken together, these data indicate that WYBQ-4 has a significant therapeutic effect against S. aureus (5 × 105 CFU) via thigh injection, followed by subcutaneous administration of WYBQ-4  treatment , the bacterial load decreased to 5.97 log10 CFU/g and 5.21 log10 CFU/g, respectively. WYBQ-4 (300 mg/kg) exhibited more prominent activity in removing bacteria from thigh tissue than moxalactam (300 mg/kg) in both the MRSA and MSSA infection groups  followed by treatment with WYBQ-4   . The sur5 mg/kg) . For the5 mg/kg)  and D. TS. aureus, which has developed multiple strategies to combat antibiotics. For example, MRSA resistance to β-lactam antibiotics is attributed to the involvement of the mecA-encoded PBP2a in cell wall synthesis, which has a low affinity for β-lactam antibiotics   , 24. Furibiotics , 26. As ibiotics –29. We f50) of 2 μg/mL, which is significantly better than that of ceftriaxone  and oxacillin , as reported (50 of WYBQ was comparable to that of ceftriaxone (1.6 μg/mL) for PBP2a , further inhibiting the transpeptidation and synthesis of the bacterial cell wall.BOCILLIN FL is able to bind to PBPs specifically and has been widely used to identify the affinity between small-molecule drugs and PBPs . Subsequreported . Furtheror PBP2a . A stronor PBP2a . SubsequS. aureus is a medically important pathogenic bacterium. It can cause extensive purulent damage involving multiple organs in humans and animals, including skin infections, pneumonia, endocarditis, toxic shock syndrome, systemic infections, and septic arthritis, and is a major source of hospital outbreaks and community-acquired infections  with constant shaking or on BHI agar plates unless otherwise noted. Escherichia coli was grown overnight in lysogeny broth  or on LB plates at 37°C. When required for selective growth of S. aureus, 10 μg/mL chloramphenicol (Cm) and erythromycin (Em) were added to the culture medium. For E. coli, 50 μg/mL kanamycin (Kan) was used for selection. WYBQ-4 is a new type of cephalosporin antibiotic with a molecular formula of C22H22N6O5S3  are presented in the Fig. S1 and S2.Detailed bacterial strain information is provided in Table S1. The clinical strains of MRSA and MSSA isolated in Beijing, Sichuan, and Shanghai in the last 3 years totaled more than 300 strains. Clinical isolates were stored at −80°C in 20% glycerol-rich brain heart infusion (BHI) and passaged twice on Trypticase soy agar prior to the experiment. Before the experiment, 16s RNA identification was performed, and then PCR was used to distinguish MRSA and MSSA by identifying the ≥8 μg/mL . FinallyS. aureus were determined using a broth microdilution method following the guidelines outlined by the Clinical and Laboratory Standards Institute (CLSI) (5 CFU of bacteria in 100 μL of cation-adjusted Mueller-Hinton broth (CAMHB) were incubated at 37°C for 16 h prior to determining the MIC. The absorbance was measured at 600 nm, and experiments were repeated in three biological replicates.The antibacterial spectrum and susceptibility of WYBQ-4 against MRSA USA300, MSSA 29213, and clinically isolated e (CLSI) , 38. Briin vitro. Overnight cultures were diluted to 106 CFU (CFU)/mL in 10 mL of fresh Mueller-Hinton broth (MHB) medium. Different amounts of antibiotics were added so that the final concentrations were equivalent to 0.5× MIC, 1× MIC, 2× MIC, 4× MIC, 8× MIC, and 16× MIC of WYBQ-4. After incubation at 37°C with shaking for 0 to 24 h, 100-μL samples from each time point were subcultured on MHB agar plates and incubated at 37°C for 24 h. MSSA 29213 and MRSA USA300 without WYBQ-4 were used as control groups. Colony numbers were counted to calculate the CFU, and the data were expressed as log10 CFU. Three biological replicates were performed for the experiment.The bactericidal activities of WYBQ-4 against MSSA 29213 and MRSA USA300 were determined S. aureus (106 CFU/mL) was supplemented with final concentrations of 2× MIC and 8× MIC of WYBQ-4. After incubation at 37°C for 1 h, WYBQ-4 was removed by centrifugation and washing with phosphate-buffered saline (PBS). Subsequently, fresh MHB medium was added to resuspend the bacteria, and the mixture was incubated at 37°C with shaking. The number of colonies was counted by taking 10 μL of bacteria at 1-h intervals and applying them to BHI agar plates after appropriate dilution. PAE duration was calculated as follows: time (h) for bacteria to return to log-phase growth after drug treatment – time (h) for the control group to return to log-phase growth (The postantibiotic effect (PAE) of WYBQ-4 against MSSA 29213 was determined in MHB agar using the viable plate count method . Brieflye growth .Cytotoxicity assay. The cytotoxicity of WYBQ-4 against A549, HEK-293T, or HepG2 cells was evaluated with a cell counting kit-8 kit  according to the manufacturer’s instructions (4 cells were seeded in 96-well plates containing prewarmed medium (100 μL) and incubated for 24 h at 37°C in 5% CO2. Then, various concentrations of WYBQ-4 (0 to 512 μg/mL) were added and further incubated for 24 h. Afterward, the cells were incubated with 100 μL of medium with 10 μL of CCK8 solution (Sigma-Aldrich) for 4 h. Finally, a solution of the purple precipitate dissolved in dimethyl sulfoxide (DMSO) was added to a 96-well plate, and the absorbance was measured at 450 nm.ructions . In brieHemolysin activity. Rabbit blood cells (RBCs) and goat blood cells (GBCs) were washed three times with sterile PBS, centrifuged  to collect the blood cells, and then resuspended in PBS to obtain an 8% blood cell suspension. The blood cells were mixed with various concentrations of WYBQ-4 ranging from 0 to 512 μg/mL (0.2% vol/vol). Triton X-100 with the same treatment was used as a control. After incubation at 37°C for 1 h, the samples were centrifuged , and the absorbance of the supernatant was measured using a microplate reader at a wavelength of 570 nm.Acute toxicity test in mice. The acute toxicity test in mice was performed according to the requirements of the Chinese Guidelines for Acute Toxicity of Chemicals (H-GPT1-1) (-GPT1-1) . In brie600) reached 0.3. After addition of 4 μg/mL (1/2 of their respective MIC) WYBQ-4, the culture was shaken at 30°C for 3 h. Bacterial cells treated with 4 μg/mL ceftaroline fosamil (1/2 of their respective MIC) served as a control group. Untreated S. aureus served as a negative control. Bacteria were collected and washed in PBS and resuspended in 4% (vol/vol) glutaraldehyde fixation overnight. The fixed samples were further processed, and morphological changes in S. aureus were observed by TEM .MRSA USA300 was cultured overnight, diluted (1:100) into 20 mL of Trypticase soy broth (TSB) medium, and incubated at 30°C with shaking until the optical density at 600 nm  and cultured in LB medium containing Kan resistance until the OD600 reached 0.6, and PBP expression was induced overnight at 16°C by adding 0.5 mM IPTG . The bacteria were then lysed by sonication, the pellet was collected, and the proteins were purified using a HIS-Select nickel affinity gel system .Using the genome of PBP binding assays were performed as previously described , 31. Selhttps://www.rcsb.org/) with PDB ID 1VQQ and a resolution of 1.80 Å. Subsequently, the protein was preprocessed using Discovery Studio software to remove water molecules, hydrogenation, and charges, extract the proligands from the structure, and visualize the processed protein using PyMOL software. WYBQ-4 is docked with 1VQQ using the active site docking mode, which is centered at center-x_29.481, center-y_28.946, and center-z_86.704. Molecular docking was performed by applying AutoDock Vina software to predict the interaction and energy between WYBQ-4 and PBP2a.The crystal structure of the PBP2a protein was obtained from the PDB database . After 2 h of infection, mice were given WYBQ-4 via subcutaneous injection and reinjected at 12-h intervals, whereas mice were treated with ceftaroline fosamil at the same doses and served as positive controls. Mice injected with only MRSA USA300 were used as a negative control. Survival of mice was recorded within 72 h, and survival rates were calculated.The animal experiments were performed using 6-week-old male C57BL/6J mice, and the mouse model of lung infection caused by MRSA USA300 was established as previously described , 44. For8 CFU of MRSA USA300 to assess bacterial load and pathological changes. The dose and manner of administration in mice were consistent with the survival experiments. In addition, the mice treated with ceftaroline fosamil at 75 and 37.5 mg/kg were used as control groups. At 48 h postinfection, mice were euthanized, and the left lungs were aseptically removed, weighed, and homogenized. Then, the lung tissues were homogenized and serially diluted for CFU counts. The right lungs were fixed with 10% formalin, embedded in paraffin, thin-sectioned, subjected to conventional hematoxylin and eosin (H&E) staining, and then observed using an optical microscope.The mice were challenged with 1 × 103). Mice were infected by intramuscular injection of 0.1 mL of S. aureus culture (5 × 105 CFU) into the right thigh. At 2 h postinfection, mice (n = 4) were treated with WYBQ-4 at single subcutaneous doses of 90 and 45 mg/kg, while mice treated with ceftaroline fosamil at the same doses served as positive controls. Then, 24 h after infection, mice were euthanized, and the right thighs were aseptically removed, weighed, homogenized, and applied to nonresistant BHI plates for CFU counting.The therapeutic effects of WYBQ-4 on thigh infections induced by MRSA USA300 and MSSA 29213 in mice  were evaluated. According to a previous description , two conS. aureus-induced systemic infection model of mice  was established as previously described (7 CFU per mouse) by intraperitoneal injection and treated with different doses of WYBQ-4, cefepime, or moxalactam at 0 and 3 h postinfection by tail intravenous injection. The mice infected in the same way were treated with physiological saline as an infection control group. The mock group was not infected with S. aureus, and the 7-day mortality of the mice in each group was calculated.The escribed . The mict tests (two-tailed) were used to compare two data sets. The log-rank test was used for survival analysis. P < 0.05 was considered statistically significant.All data are presented as the mean ± standard deviation (SD). Statistical analyses were performed using Prism software v8.0 . Student’s The animal experiments were conducted in accordance with the principles of the Basel Declaration and the guidelines of the Animal Care and Use Committee of Jilin University; the IACUC  number is 201904017.All data generated during this study are available upon request from the corresponding authors.


By establishing a rat diabetes model in rats with intervening treatment by Jiangtang Tongmai Prescription (JTTMP), this study explored the restorative pairing effect of JTTMP on diabetic lung injury. The model of type II diabetes model was used to establish the rat diabetes model, using a high-fat diet and streptozotocin (STZ) induction. Different doses of JTTMP and metformin were administered as a therapeutic to intervene, and blood was collected to assess the blood glucose level of each group of rats. HE (Hematoxylin and eosin (H&E) staining was performed to detect the morphological changes in rat lung tissue and enzyme-linked immunoassay ELISA was used to detect and quantify the expression of interleukin (IL)-6, TNF tumor necrosis factor-ɑa, and IL-1β in serum and the lung tissue of each group of rats. The level expression of TGF-β1 , Smad2, Smad3, Smad7, and other signaling pathway proteins were assessed by Western blot. In comparison with the normal control (NC) group, rats in the diabetes model (DM) group lost weight and showed significantly increased blood sugar levels. The levels of TGF-β1 and Smad2/3 were increased in the DM group but Smad7 decreased. After 8 weeks of JTTMP intervention, the level of TGF-β1 and Smad2/3 decreased but Smad7 increased, blood sugar decreased significantly and the expression of inflammatory factors in lung tissue decreased. Therefore, JTTMP may activate SnoN and the downstream TGF-β1/Smads signaling pathway to repair diabetic lung injury, which suggests its application has potential for future clinical treatment of diabetes with lung injury. Diabetes mellitus is a metabolic disease that presents with chronically increased glucose levels. Diabetes is clinically characterized by hyperglycemia, with symptoms such as polydipsia, polyuria, polyphagia, and weight loss. In addition to high blood sugar, it is often accompanied by a series of metabolic disorders related to protein, fat, water, and electrolyte processing. Without timely and effective treatment it can cause disease to multiple systems of the body, including diabetic retinopathy, nephropathy, diabetic foot, diabetic nerve disease, and more severe cases such as diabetic ketoacidosis, diabetic lactic acidosis, and diabetic nonketotic hyperosmolar coma . RecentlThe JTTMP Traditional Chinese medicine and STZ were freshly prepared in our laboratory. Metformin ; blood glucose test strips for rat interleukin (IL)-6, tumor necrosis factor (TNF)-ɑ, IL-1β factor enzyme-linked immunoassay (ELISA) detection kit ; eosin, hematoxylin, neutral resin ; Protein Marker, BCA protein concentration determination kit ; polyvinylidene fluoride membrane, enhanced chemiluminescence reagent ; Tween-20 ; RIPA tissue cell rapid lysate ; transforming growth factor (TGF)-β1, Smad2, Smad3, Smad7, p-Smad7, Ski-N terminal (SnoN), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) primary antibody ; p-Smad2 antibody ; p-Smad3 antibody ; Goat anti-Rabbit immunoglobulin G ; MaxVision TM secondary antibody and horseradish peroxidase (HRP)-Polymer .The equipment used included: Electronic balance ; blood glucose monitor; surgical straight scissors, tissue forceps ; inverted fluorescence microscope ; high-speed refrigerated centrifuge, microplate reader, plate washer ; constant temperature oven ; automatic tissue dehydrator, stall Slice baking machine ; paraffin slicer ; biological tissue embedding machine-freezer ; automatic chemiluminescence analyzer ; water bath ; and ultrapure water device .The study included 36 rats, half male and half female, 8 weeks old, and weighing about 250 g each. The animals were obtained from Three Gorges University with laboratory animal license number: SYXK (E) 2018-0104, certificate No. 42010200003097. Rats were raised under SPF conditions. The housing conditions were: temperature of 22–26°C, relative humidity of 50–60%, artificial light and dark conditions for 12 h, and adaptive feeding for 1 week. 6 rats were selected as the normal control group, and the other 30 rats were the model group. They were given high-sugar and high-fat diet  for 4 weeks : fed with ordinary feed; ② Diabetes model group (DM): equal volume of drinking water gavage. Gavaged once a day for 8 consecutive weeks and administered high-fat diet; ③ JTTMP low-dose group (DM+JTTMP 63 mg/kg): JTTMP 63 mg/mL suspension 1 ml/(kg·d) was given by intragastric administration for 8 weeks, and high-fat diet was administered; ④JTTMP middle-dose group (DM+JTTMP 126 mg/kg): JTTMP 126 mg/mL suspension 1 ml/(kg·d) was intra-gastrically administered for 8 weeks and the high-fat diet was continued; ⑤ JTTMP high-dose group (DM+JTTMP 252 mg/kg): JTTMP 252 mg/mL suspension 1 mL/(kg·d) was gavaged for 8 weeks, and high-fat diet was administered; and ⑥ Metformin group (DM+Met): 54.3 mg/ml Metformin aqueous solution of 1 ml/(kg·d) gavaged and administered for 8 weeks, and high-fat diet was administered.After the drug treatments were completed, the rats in each group were sacrificed, and the serum was frozen for later use. The left lung tissue was fixed with 4% paraformaldehyde, and the right was stored at −80°C until analysis.During the experiment, the activity, eating, drinking, and excretion of the rats were observed, and the weight of the rats was recorded every day. The comparison of weight changes in each group was statistically analyzed.Blood from the tail vein was collected in each group of rats before and after drug intervention, the fasting blood glucose was measured by the test strip method, and the blood glucose value data was recorded.The serum and lung tissue homogenate of rats from each group was collected, and ELISA was performed using 50 μL of each standard and sample solution, and phosphate-buffered saline as a negative control. An equal volume of enzyme-labeled IL-6 or TNF-α and IL-1β antibodies was added to the relevant sample and standard wells. Plates were sealed with film and incubated at 37°C for 30 min. Plates were spun and washed. The developer (50 μL) was added and incubated at 37°C for 10 min. Finally, 50 μL of stop solution was added and the absorbance  at a wavelength of 450 nm was measured. A standard curve was used to calculate the concentrations of IL-6, TNF-α, and IL-1β in the sample.The lung tissue of rats from each group was collected, fixed with 4% paraformaldehyde, and embedded in paraffin. As per routine practice, tissue sections were deparaffinized, stained with hematoxylin for 3–6 min, washed for 1–2 min, differentiated with 1% hydrochloric acid alcohol for 1–3 s, promoting liquid returned to blue for 5–10 s, washed with running water for 15–30 s, stained with 0.5% eosin solution for 2–3 min, washed with distilled water for 1–2 s, then 80% ethanol for 15–30 s, then 95% ethanol for 15–30 s, and finally anhydrous ethanol for 1–2 s. After drying, sections were sealed with neutral gum. The pathology of lung tissues such as the alveolar wall and inflammatory infiltration were analyzed under a microscope.® 20 Detergent (TBST) thrice and incubated with HRP-labeled secondary antibody (1:20000) for 2 h at room temperature. The membrane was again washed with TBST thrice. Finally, the enhanced chemiluminescence was analyzed after exposure in the darkroom.Rat lung tissues were collected from each group, briefly homogenized, and protein extraction reagent at 5 μl/mg and protease inhibitor phenylmethylsulfonyl fluoride was added at a final concentration of 1 mM. Tissues were lysed for 15 min on ice and centrifuged at 4°C, 13800 r/min for 15 min. The protein concentration of the supernatant was quantified and adjusted. Protein samples had loading buffer added and were denatured at 100°C for 5 min. Electrophoresis separation was performed by 120 g/L sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the nitrocellulose membrane was transferred, and 5% skimmed milk powder was used to block for 2 h at room temperature, then TGF-β1 antibody (1:1000), Smad2 antibody (1:1000), Smad3 antibody (1:1000), Smad7 antibody (1:1000), p-Smad2 antibody (1:1000), p-Smad3 antibody (1:1000), p-Smad7 antibody (1:1000), and GAPDH antibody (1:1000) were incubated overnight on samples at 4°C. The membrane was washed with tris-buffered saline with 0.1% Tweent-test for the comparison of two sample means was performed. Data were expressed as the P<0.05 indicates that the difference was statistically significant.All data were analyzed by SPSS 21.0. The P<0.01). Compared with the DM group, the extent of weight loss in the DM+JTTMP 126 mg/kg group was reduced (P<0.05), and the weight loss in the DM+JTTMP 252 mg/kg and the DM+Met groups were significantly improved. In summary, the weight of the DM+Met group at each time point was much higher than that of the DM group . The glucose level in the DM group remained higher than in the NC group after 8 weeks (P<0.001). There was no obvious difference in blood glucose between the DM group and the treated group before the drug intervention; however, for 8 weeks after the drug intervention, the glucose in the DM+JTTMP 126 mg/kg group decreased to be lower than the DM group (P<0.05). The blood glucose level in the DM+JTTMP 252 mg/kg and DM+Met groups further decreased to be significantly lower than that of the DM group  was higher than that of the NC group . The level of inflammatory factors in the DM+JTTMP and DM+Met groups was significantly lower than those in the DM group (P<0.05), and as the dose of JTTMP increased, the expression of inflammatory factors gradually decreased. In the high-dose group with JTTMP 252 mg/kg, inflammatory factors were significantly reduced (P<0.001) to similar levels close to the normal level  but was increased in the DM+JTTMP and DM+Met groups after treatment , Wuhan Health and Family Planning Commission Key Scientific Research Project (WZ20A06), and Wuhan Young and middle-aged Medical Talents Training Project ([2013]35).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.