Poly(acrylonitrile) (PAN) fibers have two essential drawbacks: they are usually processed by solution-spinning, which is inferior to melt spinning in terms of productivity and costs, and they are flammable in air. Here, we report on the synthesis and melt-spinning of an intrinsically flame-retardant PAN-copolymer with phosphorus-containing dimethylphosphonomethyl acrylate (DPA) as primary comonomer. Furthermore, the copolymerization parameters of the aqueous suspension polymerization of acrylonitrile (AN) and DPA were determined applying both the Fineman and Ross and Kelen and Tüdõs methods. For flame retardancy and melt-spinning tests, multiple PAN copolymers with different amounts of DPA and, in some cases, methyl acrylate (MA) have been synthesized. One of the synthesized PAN-copolymers has been melt-spun with propylene carbonate (PC) as plasticizer; the resulting PAN-fibers had a tenacity of 195 ± 40 MPa and a Young’s modulus of 5.2 ± 0.7 GPa. The flame-retardant properties have been determined by Limiting Oxygen Index (LOI) flame tests. The LOI value of the melt-spinnable PAN was 25.1; it therefore meets the flame retardancy criteria for many applications. In short, the reported method shows that the disadvantage of high comonomer content necessary for flame retardation can be turned into an advantage by enabling melt spinning. Poly(acrylonitrile) (PAN) used for textile fibers is mostly polymerized by precipitation polymerization and then solution-spun from polar solvents. PAN-based fibers are widely used for clothing, home textiles and technical textiles ,4,5,6,7.PAN has a “Limiting Oxygen Index” (LOI) of about 17–18 determined according to DIN EN ISO 4589-1, i.e., it starts to burn at oxygen concentrations of the surrounding atmosphere ≥17–18 vol% , if it is exposed to a flame for longer than a second ,11.To increase the LOI of a polymer, one has to add flame retardants. These can be introduced as additives, as comonomers, by chemical modification or by coating. Additives and coatings are usually the cheapest way to provide a fiber with flame retardant properties  however,The most powerful chemical modification known for PAN is a post-spinning-treatment with hydrazine ,14,15,162, PO, HPO) suppress the radical chain reactions by recombination [3) [An example for flame retardant comonomers of acrylonitrile are halogenated comonomers like vinyl chloride, vinylidene chloride and vinyl bromide. They are used in concentrations of 30–50 mol%. The resulting intrinsically flame-retardant fibers reach LOI values of up to 30 and are known as “modacrylic fibers” ,20. Howebination ,32. In ttion [3) .A promising phosphorus-containing comonomer for PAN is dimethylphosphonomethyl acrylate (DPA) (16.0 wt% P), which has already been proven to copolymerize readily with acrylonitrile (AN) and allow for wet-spinning, resulting in fibers with LOI values of ≥ 25 at ca. 3 wt% phosphorus . In caseAcrylonitrile (>99%), acryloyl chloride (96%), dichloromethane (99.9%), methanol (99%), methyl acrylate (>99%), potassium carbonate (99%), sodium persulfate (98%), and triethyl amine (99%) were purchased from abcr GmbH . Sodium metabisulfite (99%), propylene carbonate (99%), iron (II) sulfate hydrate (>99%), and aluminum sulfate hydrate (98–102%) were purchased from Alfa Aesar . Sulfuric acid (96%) was purchased by Acros Organics . Dimethyl phosphite (98%) was purchased from Sigma Aldrich . All chemicals were used as received. 2CO3 (6.77 g 0.05 mol) was then added to the stirred mixture. The mixture was allowed to warm to room temperature and was stirred for four hours. The K2CO3 was then filtered off and the volatile components were removed at reduced pressure (1 ∙ 10−2 mbar). Yield: 98 g, 70%. 1H-NMR  δ = 4.28 , 3.91 , 3.77  ppm. 31P-NMR  δ = 26.9 ppm. 13C-NMR  δ = 56.1 , 53.2  ppm. HRMS (ESI): m/z calculated for C3H10O4P+: 141.0322. Found: 141.0319.Dimethyl (hydroxymethl)phosphonate was synthesized according to El Asri et al.  Dimethyl2Cl2 (400 mL) and the solution was cooled to 0 °C. Within one hour, acryloyl chloride  dissolved in 100 mL CH2Cl2 was added. The reaction mixture was stirred for another two hours. The resulting triethylamine hydrochloride was filtered off and all volatile components were removed under reduced pressure. The raw product distilled at 83 °C and 1.7 ∙ 10−2 mbar. Yield: 40 g, 71%. 1H-NMR : δ = 6.49 , 6.18 , 5.93 , 4.49 , 3.82  ppm  δ = 21.57 (s) ppm  δ = 165.0 , 132.5, 127.0, 56.0 , 53.3  ppm. HRMS (ESI): m/z calculated for C6H12O5P+: 195.0428. Found: 195.0417.Dimethyl phosphonomethylacrylate was synthesized according to Liepins et al.  Dimethyl 6H) ppm . 31P-NMR (s) ppm . 13C-NMRAqueous precipitation polymerizations were carried out under an argon atmosphere at 56 °C in aqueous, acidic medium (0.03 molar aqueous sulfuric acid solution). After preheating the aqueous medium, the monomer mixture was added and stirred for one minute. The monomer concentration in the aqueous reaction mixture was 6.7 wt%. Then, the initiator was added. The initiator system consisted of sodium persulfate , sodium metabisulfite , catalytic amounts of iron (II) sulfate  and aluminum sulfate  dissolved in demineralized water . After adding the initiator to the stirred reaction mixture, the polymerization mixture was stirred for another hour. The polymer precipitated as a fine white powder which was filtered off, washed twice with water and methanol, respectively, and dried at 80 °C under atmospheric pressure. The resulting polymers and their properties are summarized in G and H, which are being used in the graphic methods to determine the copolymerization parameters according to Fineman and Ross [The parameters and Ross  and Keleand Ross ,47 were DPAM and ANM are the molar amounts of DPA and AN in the educt mixture and DPAm and ANm are the molar amounts of AN and DPA in the copolymer. In the Fineman and Ross plot, the parameters G and H are plotted linearly according to Equation (3) to determine DPAr and ANr:In these equations, G and H are plotted according to Equation (4):For the inverted Fineman and Ross plot, α to distribute the data points evenly (Equation (5)):Kelen and Tüdõs extended the Fineman and Ross method with a parameter In addition to drying at 80 °C under atmospheric pressure after polymerization, all polymers were dried under vacuum at 100 °C for 5 h prior to rheology measurements, spinning trials, and film preparation in a vacutherm VT 6130 M vacuum furnace  equipped with a rz-16 vacuum pump . The residual water content was determined by Karl Fischer titration, which was performed on an 899 Coulometer and an 885 Compact Oven SC  at 140 °C. The resulting water content was <0.1 wt%.PAN films were prepared by doctor blading and the immersion casting technique. First, a 24 wt% solution in DMSO was prepared for each PAN. Then, the viscous polymer solution was poured onto a glass plate and evenly distributed by a stainless-steel thin film applicator (doctor blade) with a gap height of 500 μm. The glass plate was then immersed in water as a coagulation bath. After 14 h, the coagulated polymer film was removed and dried at 80 °C for 2 h. Then, they were compressed by a heat press at 6 bar pressure and 90 °C. An example of the resulting polymer film is shown in Fibers were melt-spun using a Haake MiniLab II twin-screw extruder  with a capacity of ca. 7 mL. A monofilament spinning nozzle with a diameter of 0.5 mm was used. The fibers were wound on a Sahm winder .1H-NMR.Removal of propylene carbonate (PC) was achieved discontinuously by winding the fibers around a glass vial and then putting them in stirred demineralized water at 90 °C for 5 min. The residual PC-content was below 0.1 wt% determined by 1H-NMR: 400 MHz, 13C-NMR: 101 MHz; external standard: tetramethylsilane). Spectra were analyzed using the MestReNova program . Rheology was measured on a Physica MCR 501 . Measurements were carried out with two parallel plates in a distance of 1 mm. For mechanical testing of the fibers, 20 single filament measurements of each sample were conducted on a Favimat tensiometer . The clamping length was 25 mm, the test speed was 20 mm/min. Size exclusion chromatography (SEC) measurements were performed on a 1260 Infinity device from Agilent Technologies  equipped with a Multi-Detector-Suite viscosimetry and refractive index detector. A 50 mm precolumn and a 300 mixed B column (Agilent Technologies) were used as stationary phases, DMAc containing 5 g/L LiBr was used as mobile phase. The column temperature was set to 50 °C, the flow rate was 0.75 L/min, the sample concentration was 2 mg/mL. Chromatograms were interpreted using conventional calibration against poly(methyl methacrylate) polymer standards in a molecular weight range of 102 < nM < 392,000 g/mol. Differential scanning calorimetry (DSC) measurements were carried out under air flow (20 mL/min) on a Q2000 differential scanning calorimeter  applying a heating rate of 10 K/min. The sample mass was 2 mg. High resolution mass spectrometry was carried out on a Microtof Q mass spectrometer . Elemental analysis for determining the phosphorus content was achieved via precipitation titration. 30 mg of a sample were dissolved in 10 mL of perchloric acid at 100 °C over two days. The solution was then titrated with aqueous lanthanum nitrate, resulting in the precipitation of lanthanum phosphate. A color change indicated the end of the titration, the phosphorus content was calculated from the consumed lanthanum nitrate. Limiting oxygen index (LOI) analyses were performed by burning PAN-films in a testing atmosphere with varying oxygen content in accordance to DIN EN ISO 4589-1 on an apparatus provided by Fire Testing Technology . A section of the film (5 cm × 15 cm) was used as the test specimen and clamped vertically in a holder. This holder was located inside a glass cylinder through which an oxygen-nitrogen mixture with a defined oxygen content was passed. The sample was ignited from above. By varying the oxygen content, the maximum oxygen concentration was determined at which the sample still extinguished by itself, which is the LOI.NMR spectra were measured on a Ultrashield 400 Plus instrument  at 303 K. , the molar amount of DPA required to obtain a melt-spinnable PAN was anticipated to be higher than for copolymers prepared from comonomers with copolymerization parameters with AN closer to unity, e.g., MA  [Next, five PAN-based copolymers, PAN F1–F5  were syn0.7–1.5) ,48,49,500.7–1.5) . Unfortu2r usually ca. 0.4–0.7) [Flame retardant properties can be determined by various standardized methods like the UL94 vertical flame test (DIN EN 60695-11-10), by microscale combustion calorimetry (ASTM D7309), or by the LOI vertical flame test (DIN EN ISO 4589-1). These different measurements all have advantages and limitations. They also suffer from poor intercorrelation amongst each other (0.4–0.7) . This haPAN F1, PAN F2 and PAN F5 matched the targeted phosphorus content of > 3 wt% . In ordegT and degT values were determined by DSC in air; measurements are shown in gT was in the expected range of ca. 100 °C for all PANs. The onset temperature degT of the exothermal cyclization and dehydrogenation reactions of PAN was in the range of 239–255 °C in air for PAN F1–F5 and lower than degT of a PAN-homopolymer (degT = 298 °C) [degT = 291 °C) [Additionally, both the  298 °C)  or a com 291 °C) . In this work, propylene carbonate (PC) was used as an external plasticizer in addition to DPA and MA as internal plasticizers, since PC is cheap, non-toxic and can be easily removed by washing with water . Table 5η*| > 2000 Pa s was aimed for, ideally in the range of 200–1000 Pa s. Furthermore, the loss factor tan δ should be at least ≥ 1, indicating acceptable viscoelastic behavior of the PAN/PC mixture. While PAN F1–F4 were all outside the desired |η*| < 2000 Pa s and tan δ ≥ 1, PAN F5 met the criteria. Its temperature sweep measurement is shown in η*| of was <2000 Pa s in a temperature range of 170–190 °C. Its tan δ at 170 °C was 1 and decreased slightly to 0.97 at 175 °C. This can tentatively be attributed to minor PAN-degradation over time that overlays the measurement due to the slow heating rate of 1 K/min and the comparably low degT of 239 °C. Oscillatory rheology temperature sweeps from 160 to 190 °C were measured. For melt spinning, a target viscosity of |tan δ-value, winding speeds up to 30 m/min were reached. The mechanical properties of the fiber before and after removal of the PC are summarized in Melt-spinning of PAN F5 blended with 22.5 wt% PC was successfully carried out at 175 °C. A photograph of the resulting fibers is shown in Although the fibers had a comparably large diameter due to the low winding speeds, their tenacity after washing was almost 200 MPa, which is better than expected for such low speeds. Tenacity was approx. 10–50% lower than in commercial, flammable textile PAN-fibers, but comparable to hydrazine-modified flame-retardant PAN-fibers or modacrylic fibers ,53,54. CThe scope of this work was to prepare a flame retardant, melt-spinnable PAN. This was achieved with a flame-retardant PAN-terpolymer containing both, MA and DPA, as comonomers. The copolymer was melt-spun using PC as an external plasticizer, yielding fibers with tenacities slightly lower than in commercial PAN-fibers. In agreement with the literature, PAN-copolymers with >3 wt% phosphorus incorporated had a LOI value >25.DPAr = 0.84 ± 0.05 and ANr = 3.4 ± 1.2, which indicates that a gradient in polymer composition is formed over the polymerization time.Copolymerization parameters of AN and DPA were determined to


The structures of these compounds were determined using NMR, HRESIMS, and electronic circular dichroism (ECD) data. The cytotoxicity of compounds 1–5 and their ability to inhibit nitric oxide (NO) production, as well as protect against the corticosterone-induced apoptosis of Pheochromocytoma (PC12) cells, were evaluated in vitro using the appropriate bioassays. Compounds 1 and 2 significantly inhibited the corticosterone-induced apoptosis of PC12 cells at a concentration of 20 μΜ.Five new cyclic diarylheptanoids (platycary A–E, compounds  RAW264.1–5 on corticosterone-induced apoptosis were evaluated using a previously reported method [2 at 37 °C. Poorly differentiated PC12 cells were divided into the following groups: untreated, CORT (150 μmol/L), CORT (150 μmol/L) plus DIM (10 μmol/L) as the positive control, and CORT (150 μmol/L) plus one of the test compounds (20 μmol/L). Poorly differentiated PC12 cells were seeded into 96-well culture plates at a density of 1 × 104 cells/well. After a 24 h culture, the wells were added as compounds, as previously described. Next, 48 h later, MTS solution was added to each well. An absorbance was measured at 492 nm using a Thermo Multiskan FC. All data was expressed as mean ± SD and paired t-tests implemented in SPSS 13.0 were used to evaluate the statistical significance of the differences between separate treatment groups and the control.The protective effects of compounds d method . BrieflyThe above biological activities tests were conducted at the Natural Drug Activity Screening Center, Kunming Institute of Botany, Chinese Academy of Sciences.P. strobilacea afforded five new cyclic diarylheptanoids ) and three known ones: phttyearynol (compound 6), myricatomentogenin (compound 7), and juglanin D (compound 8). Biological activities of compounds 1–5 were evaluated using cytotoxicity, NO, and corticosterone-induced apoptosis assays. Compounds 1 and 2 exhibited significant protective effects against the corticosterone-induced apoptosis of PC12 cells at the concentration of 20 μΜ, indicating that compounds 1 and 2 are two potential cytoprotective agents for neurological diseases. The identification of these new compounds significantly increases both the number of diarylheptanoids produced by P. strobilacea and the number of diarylheptanoids with potential medical value.Phytochemical investigation of the stem bark of


An integrated study on the effect of renal diet on mineral metabolism, fibroblast growth factor 23 (FGF-23), total antioxidant capacity, and inflammatory markers has not been performed previously. In this study, we evaluated the effects of renal diet on mineral metabolism, oxidative stress and inflammation in dogs with stage 3 or 4 of chronic kidney disease (CKD). Body condition score (BCS), muscle condition score (MCS), serum biochemical profile, ionized calcium (i-Ca), total calcium (t-Ca), phosphorus (P), urea, creatinine, parathyroid hormone (PTH), FGF-23, interleukin 6 (IL-6), interleukin 10 (IL-10), tumor necrosis factor alpha (TNF-α) and total antioxidant capacity (TAC) were measured at baseline (T0) and after 6 months of dietary treatment (T6). Serum urea, P, t-Ca, i-Ca, PTH, FGF-23, IL-6, IL-10, TNF-α and TAC measurements did not differ between T0 and T6. Serum creatinine (SCr) was increased at T6 and serum PTH concentrations were positively correlated with serum SCr and urea. i-Ca was negatively correlated with urea and serum phosphorus was positively correlated with FGF-23. Urea and creatinine were positively correlated. The combination of renal diet and support treatment over 6 months in dogs with CKD stage 3 or 4 was effective in controlling uremia, acid–base balance, blood pressure, total antioxidant capacity, and inflammatory cytokine levels and in maintaining BCS and MCS. Chronic kidney disease (CKD) is considered the most common renal disease in dogs . Through2 blockers (ranitidine) every 8 or 12 h and sodium bicarbonate according to blood gas results. Two animals required 0.5–1 mg/kg BW appetite stimulants (cyproheptadine hydrochloride) every 12 h, and five animals were treated with 30–60 mg/kg BW/day phosphorus binder . One animal had an episode of acute decompensation of CKD and received medical treatment. Dogs were also closely monitored to detect dehydration and no parenteral fluid administration was needed, excepted for that dog that had acute renal dysfunction. Free access of tap water was assured.Ten client-owned dogs were enrolled with a mean ± SD body weight of 16.33 ±14.64 kg, a mean age of 8.89 ± 4.46 years, various breeds and seven out of 10 were females and three of them were sterilized. Diagnosis of CKD was based on persistent azotemia over 3 months as well as imaging (ultrasound) findings of chronic kidney abnormalities. CKD classification was according to IRIS and 80% of CKD dogs were in stage 3 at T0 . All anip = 0.045). However, BCS (T0: 5.70 ± 0.67 versus T6: 5.30 ± 1.05) and MCS (T0: 2.20 ± 0.42 versus T6: 2.40 ± 0.51) showed no significant differences  between T0 and T6.A reduction in body weight was observed between T0 (16.34 ± 14.64 kg) and T6 . No differences in urine specific gravity (USG) were found between the two time points, showing isosthenuria at both times (p = 0.384)  . Proteinp = 0.853) between T0 and T6 (138.4 ± 5.49 mmHg) (Systolic blood pressure was within the reference range  and show49 mmHg) .p = 0.704) and blood bicarbonate concentration (p = 0.072) showed no differences between the two time points and were within the reference range (Blood pH (ce range .p = 0.0350). Sodium , potassium , alanine aminotransferase (ALT)  and alkaline phosphatase (ALP)  were within the reference ranges at both time points. Moreover, serum total protein  and albumin  did not differ between T0 and T6 . The packed cell volume (PCV) showed a decrease between T0 (35.3 ± 6.83%) and T6  and was below reference values at both points (RI 37–55%). Notably, serum creatinine (SCr) was increased at T6  and serum cholesterol were slightly elevated at T0 and T6 , but only cholesterol was significantly different between assessment periods  and urea , and i-Ca had a negative correlation with urea . Urea and SCr were positively correlated , and FGF-23 was positively correlated with phosphorus .PTH concentration was positively correlated with creatinine , interleukin 10 (IL-10), and tumor necrosis factor alpha (TNF-α) and TAC showed no difference between T0 and T6  analysis, as well as different treatments that had to be initiated to control clinical and laboratory alterations. The best delineation would be the inclusion of a group of animals with CKD fed with a maintenance diet. However, the use of a maintenance diet in this situation may reduce the life expectancy of the dogs included in the control group. In advanced stages of CKD, finding animals with stable renal function and no appetite changes is difficult.In conclusion, feeding dogs with advanced CKD with a renal diet for 6 months in combination with support treatment was effective in controlling uremia, acid–base balance, blood pressure, antioxidant capacity, and production of inflammatory cytokines, as well as in the maintenance of BCS and MCS. Further studies are needed to better explore how a renal diet can improve the quality of life in CKD dogs.This study was conducted at the Veterinary Hospital of the School of Veterinary Medicine and Animal Science of the University of São Paulo (FMVZ/USP), São Paulo—SP, Brazil and approved by the Ethics Committee of the Veterinary Medicine and Animal Science School of the University of São Paulo (FMVZ/USP) on 4 September 2013, protocol number 3138/2013. The research was a prospective, 6-month longitudinal dietary trial utilizing a before (T0) and after (T6) design. Ten client-owned dogs with CKD at stage 3 or 4  were incNutritional contents of the diet were balanced and met all requirements for the maintenance of adult dogs ,63; howe® Model 811-B, Oregon, United States). Blood pressure was evaluated before any clinical examination or sample collection was performed. Six measurements were performed at each time point, and the mean value was recorded. The BCS was determined according to a nine-point scale . For analysis of PTH, FGF-23, cytokines and TAC, blood samples were collected and immediately placed on ice and centrifuged at 4 °C later. Serum aliquots of 1 mL were stored at −80 °C. Urine samples were obtained by cystocentesis for urinalysis , USG (by refractometry method), urine culture and sediment microscopic examination. Blood pH and bicarbonate measurements were performed using a blood gas analyzer —b; blood2+ is reduced to Cu+, and the resulting Cu+ forms colorful complexes. The color intensity at 570 nm is proportional to the amount of TAC in the sample, and the antioxidant concentrations were expressed in μm Trolox equivalents. FGF-23 was analyzed with a human-specific FGF-23 ELISA , the values at T0 and T6 were used for the statistical analyses. The data are expressed as the mean ± SD.Statistical analyses were performed using statistical software—Statistical Analysis System (SAS) —h (Stati


Bacterial nanocellulose (BNC) membranes have enormous potential as systems for topical drug delivery due to their intrinsic biocompatibility and three-dimensional nanoporous structure, which can house all kinds of active pharmaceutical ingredients (APIs). Thus, the present study investigated the long-term storage stability of BNC membranes loaded with both hydrophilic and lipophilic APIs, namely, caffeine, lidocaine, ibuprofen and diclofenac. The storage stability was evaluated under accelerated testing conditions at different temperatures and relative humidity (RH), i.e., 75% RH/40 °C, 60% RH/25 °C and 0% RH/40 °C. All systems were quite stable under these storage conditions with no significant structural and morphological changes or variations in the drug release profile. The only difference observed was in the moisture-uptake, which increased with RH due to the hydrophilic nature of BNC. Furthermore, the caffeine-loaded BNC membrane was selected for in vivo cutaneous compatibility studies, where patches were applied in the volar forearm of twenty volunteers for 24 h. The cutaneous responses were assessed by non-invasive measurements and the tests revealed good compatibility for caffeine-loaded BNC membranes. These results highlight the good storage stability of the API-loaded BNC membranes and their cutaneous compatibility, which confirms the real potential of these dermal delivery systems. Komagataeibacter genus (formerly known as the Gluconacetobacter genus) [Cellulose has been used throughout the years as a substrate for the design of functional blends, composites and hybrid materials in various fields of application ,2,3. Addr genus) ,10.® Dura Repair , Epicitehydro  and Celmat , which are FDA approved and/or CE-certified for biomedical applications [In the last decade, bacterial exopolysaccharide (viz. BNC) has attracted a great deal of interest in several areas, ranging from nanocomposite materials ,12,13 toications . Moreoveications . Severalications ,23, ibupications , diclofeications  and amoxications  in theirications ,28 for cications  and BNC-ications .Despite good in vivo skin compatibility  and the −1 [−1 [N- acetamide) is a hydrophilic local anesthetic drug with water solubility of 4.1 mg mL−1 [−1 [−1 [−1 [−1 [In the present study, BNC membranes were individually loaded with different active pharmaceutical ingredients (APIs), namely, caffeine, lidocaine (in the hydrochloride hydrate form), ibuprofen and diclofenac  A. These −1 ) and eth−1 [−1 ); lidoca mg mL−1 ; ibuprofmL−1 [−1 ) and poo [−1 [−1 ); and fi [−1 [−1 ) but wit [−1 [−1 ).2 of membrane) was added as a plasticizer to increase the malleability and conformability of the membranes, in view of their cutaneous applications [2 of membrane). In terms of composition  in the case of caffeine) and ibuprofen in absolute ethanol, into the wet BNC network A accordiications ,23,24,25position , and sinulations ,23,24,25−1 allocated to the O–H stretching vibration of the primary and secondary hydroxy groups, 2898 cm−1 assigned to the stretching vibration of the C–H bonds, 1315 cm−1 ascribed to the O–H in the plane bending vibration of the primary and secondary hydroxy groups, 1160 cm−1 attributed to the C–O–C antisymmetric stretching vibration of the glycosidic bonds, and 1031 cm−1 assigned to the C–O stretching vibration [−1 (O–H stretching), 1100 cm−1 and 1030 cm−1  [All API-loaded BNC membranes and their individual components were characterized by Fourier transform infrared-attenuated total reflection (FTIR-ATR) spectroscopy. ibration . The abslcohols) , are ove−1 (C-H stretching from methyl groups), 1690–1428 cm−1  and 1300–1000 cm−1 (C-N and C-C bond vibrations); (ii) lidocaine: 3250 cm−1 (N-H bonds of secondary amide), 1650 cm−1 (C=O bonds of the amide groups) and 1550 cm−1 (aromatic C=C bonds); (iii) ibuprofen: 3000 and 2750 cm−1 (CH2 and CH3 stretching), 1705 cm−1 (C=O stretching), 1507 cm−1 (aromatic C=C bonds), 936 cm−1 (CH3 rocking vibration) and 668 cm−1 (C-H out of plane deformation) [−1 (N-H stretching), 1603 cm−1 , 1572 cm−1 , 1350–1250 cm−1 (C-N stretching), and 730–745 cm−1  [On the other hand, the spectra of the APIs exhibit their typical vibrations B as follrmation) ; and (ivd rings) . All figd rings) ,23,24,252 of membrane (The morphology of the API-loaded BNC membranes was assessed by scanning electron microscopy (SEM) and the surface and cross-sectional micrographs are presented in membrane . These mAccording to the WHO Technical Report Series , environThe simple observation of the API-loaded BNC membranes B evidencSince these API-loaded BNC membranes were submitted to different levels of environmental humidity, it is relevant to measure their moisture-uptake after three months under different storage conditions. Overall, the moisture-uptake increased with increasing RH (from 60% to 75%) for all membrane samples . While BRegarding the API-loaded BNC membranes, the moisture-uptake is dependent on the API nature with lidocaine and diclofenac exhibiting the highest moisture-uptake values particularly at higher RH and temperature, namely, 19.8 ± 4.1% at 60% RH/25 °C and 36.3 ± 2.1% at 75% RH/40 °C for BNC/lidocaine, and 9.2 ± 1.0% at 60% RH/25 °C and 31.3 ± 7.3% at 75% RH/40 °C for BNC/diclofenac . These dWhen the storage conditions were changed to 0% RH at 40 °C for three months, all samples exhibited weight-loss, most likely due to the removal of adsorbed water and glycerol. It is evident that the incorporation of the APIs into the plasticized BNC membranes reduced their weight-loss values from 27.0 ± 8.0% for the BNC plasticized with 1% (w/v) of glycerol to a minimum of 5.2 ± 0.6% for BNC/ibuprofen, and also from 37.2 ± 10.1% for the BNC containing 5% (w/v) glycerol to 25.4 ± 0.7% for BNC/diclofenac .In order to investigate possible interactions and/or degradation phenomena occurring after exposing the API-loaded membranes to distinct storage conditions, their structure was once again studied by FTIR-ATR . The speThe morphology of the API-loaded BNC membranes was again verified by SEM after storing the membranes at 75% RH/40 °C, 60% RH/25 °C and 0% RH/40 °C for three months in the dark, as shown in Despite the absence of visible changes at the structural and morphological level, it is necessary to confirm that the in vitro release profiles of the API-loaded BNC membranes remain the same after three months under different storage conditions. These assays were carried out in PBS at pH 7.4 to simulate the pH of the skin, before and after storing the membranes at 75% RH/40 °C, 60% RH/25 °C and 0% RH/40 °C, as summarized in −1 [−1 [−1 [−1 [Furthermore, all membranes achieved a total release (100%) of the respective API after 5 min in the case of caffeine and lidocaine, 30 min for ibuprofen and 15 min for diclofenac . These d−1 ) and lid−1 [−1 ) are the [−1 [−1 ) and ibu [−1 [−1 ). Theref [−1 [−1 ,24,25. T [−1 [−1  and dicln) of 0.52 (regression coefficient: R2 = 0.999) was achieved for BNC/caffeine, 0.91 (R2 = 0.988) for BNC/lidocaine, 0.48 (R2 = 0.998) for BNC/ibuprofen and 0.55 (R2 = 0.999) for BNC/diclofenac. These fitting parameters are indicative of Fickian diffusion  in the case of BNC/ibuprofen, and non-Fickian transport  in the case of BNC/caffeine, BNC/lidocaine and BNC/diclofenac [In addition, the initial drug release profiles of the API-loaded BNC membranes can be fitted to the Korsmeyer–Peppas kinetic model ,46 accorechanism ,47. Accoclofenac ,46,47.A look at the drug release profiles after exposure to the accelerated testing conditions shows that the BNC/diclofenac membrane experienced the smallest effects with almost no difference in the amount of API liberated as a function of time. The other three API-loaded BNC membrane systems experienced some changes that resulted in faster release rates. For example, after storing the membranes at 75% RH/40 °C for three months in the dark, the release of (i) caffeine increased from 78 ± 3% to 90 ± 1% after 2 min, (ii) lidocaine increased from 62 ± 10% to 80 ± 2% after 2 min, and (iii) ibuprofen increased from 32 ± 1% to 45 ± 7% after 2 min. These differences can probably be attributed to a higher API content at the surface of the membranes due to migration during storage followed by precipitation at the surface. Lastly, the fitting parameters to the Korsmeyer—Peppas kinetic model  were alsIt is worth noting that the rapid release profile that was observed is suitable for the therapeutic effect intended for the APIs used in this study. In order to achieve a rapid local anesthetic, analgesic and anti-inflammatory action, a fast-transcutaneous penetration must be ensured, which is only possible if a sufficient amount of drug is released from the delivery system. This scenario is also applicable in the case of caffeine, where the API has to reach the innermost layer of the skin, the adipose tissue.BNC is known for exhibiting in vitro compatibility with epidermal cells , butIn the visual assessment, both plasticized BNC and caffeine-loaded BNC membranes performed well in the compatibility tests with all volunteers scoring zero cutaneous responses at every test site according to the International Contact Dermatitis Research Group (ICDRG) scale . StartinRegarding the plasticized BNC membrane, it was very clear that this exopolysaccharide did not promote any skin irritation a*, B, comprop < 0.05) from 0.92 ± 0.17 for the control to 1.41 ± 0.22 for BNC/caffeine. This effect is also attributable to the moisturizing effect of glycerol, which has a dual effect in topical delivery systems, namely, plasticizer and moisturizer.As in the case of the plasticized BNC membrane, the non-invasive measurements for the caffeine-loaded BNC membrane established that it did not cause any alterations of the skin at any of the tested sites. The basal values of skin redness (a*) were preserved after membrane application, which indicates that it did not cause any erythema, viz. no skin irritation B. Furthe® . Ultrapure water  at 0.5 L min−1) was purified by a Simplicity® Water Purification System . All other chemicals and solvents were of laboratory grade.Citric acid (≥99.5%), glucose (≥99.5%), potassium sulphate (≥99.0%), sodium phosphate dibasic (≥99.0%), caffeine (99%), ibuprofen (≥98%), lidocaine hydrochloride monohydrate (99.5%), diclofenac sodium salt (≥98.5%) and glycerol (≥99.5%) were purchased from Sigma-Aldrich . Bacteriological agar, peptone and yeast extract were acquired from Himedia Laboratories GmbH . Phosphate buffer saline  was supplied from GibcoGluconacetobacter sacchari bacterial strain [−1 glucose, 5 g L−1 peptone, 5 g L−1 yeast extract, 2.7 g L−1 Na2HPO4, 1.15 g L−1 citric acid, and 15 g L−1 agar, pH 5) under static conditions. After incubation for several days at 30 °C, the BNC membranes were separated from the media, treated with 0.5 M NaOH aqueous solution and repeatedly washed with ultrapure water to remove culture media components. Lastly, the membranes were whitened with 1% sodium hypochlorite solution, washed several times with water until neutral pH, and stored in ultrapure water in the refrigerator until further use.Wet membranes of bacterial nanocellulose (BNC) characterized by a three-dimensional network of nano- and microfibrils, were biotechnologically produced in our laboratory using the l strain . BrieflyBNC membrane discs (diameter: ca. 7 cm) were individually loaded with different APIs, namely, caffeine, lidocaine, ibuprofen and diclofenac, as described in other studies ,23,24,25The entrapment efficiency of each API-loaded BNC membrane was calculated as follows:The drug loading of each API-loaded BNC membrane was determined in terms of API mass per area of BNC membrane, according to the following equation:−1 at a resolution of 4 cm−1 over 32 scans in a Perkin-Elmer FT-IR System Spectrum BX spectrophotometer  equipped with a single horizontal Golden Gate ATR cell.Fourier transform infrared-attenuated total reflection (FTIR-ATR) spectra were collected in the range of 600–4000 cmSurface and cross-section micrographs of the membranes were obtained by an ultra-high-resolution field-emission SEM Hitachi SU-70 microscope  operating at 4 kV. The membranes for surface and cross-section (fractured in liquid nitrogen) examination were placed on a steel plate and coated with a carbon film prior to analysis.2) of each API-loaded BNC membrane were placed in glass vials and stored for 3 months in the dark  at different relative humidity (RH) and temperature conditions. So, the membranes were tested at 0% RH (using a desiccator with silica gel) and 40 °C, 60% RH  to ensure constant humidity throughout the study. The assay was conducted in triplicate. All samples were weighed before and after storage. The moisture-uptake of the membrane samples stored at 75% and 60% RH was calculated by the equation:Samples (1 × 1 cmsolution ) and 25 solution ) and 40 Before the drug release studies, the stored membranes were equilibrated in a desiccator filled with silica gel for 24 h. Drug release studies were performed in 200 mL of PBS, pH 7.4 at 32 °C , and theTwenty human volunteers  participated in this study, after informed oral and written consent. The study was approved by the local Ethics Committee of the Faculty of Health Sciences of the Lusófona University  and was performed in accordance with the ethical standards as laid down in the 1964 Declaration of Helsinki and its later amendments .2, Finn Chambers®, Epitest Ltd Oy, Tuusula, Finland) were applied in the volar forearm of the volunteers for 24 h. Each patch had 4 chambers containing a caffeine-loaded BNC membrane, a pure BNC membrane, distilled water (negative control) and a 2% (w/v) caffeine aqueous solution. A visual assessment of each application site was conducted 2 h after the patch removal and cutaneous responses were classified according to the International Contact Dermatitis Research Group criteria [Epicutaneous patches  and Tukey’s test  was used to determine the statistical significance established at The long-term storage stability of BNC membranes loaded with different APIs, namely, caffeine, lidocaine, ibuprofen and diclofenac was investigated. The storage stability was assessed under accelerated testing conditions at different temperatures and relative humidity (RH). All systems were stable under the studied storage conditions with no substantial structural and morphological changes or variations in the drug release profile. The only change was in the moisture-uptake, which increased with RH due to the hydrophilic nature of BNC. Moreover, the caffeine-loaded BNC membrane was chosen for in vivo cutaneous compatibility studies, where patches were applied in the volar forearm of twenty volunteers for 24 h. The cutaneous responses were measured by non-invasive methodologies and the in vivo tests showed good compatibility for caffeine-loaded BNC membranes. These results highlight the good storage stability of the APIs-loaded BNC membranes and validate the actual potential of these topical delivery systems with different APIs.


Xanthomonas oryzae pv. oryzae (Xoo) is one of the most devastating bacterial diseases of rice in temperate and tropical regions. Breeding and deployment of resistant cultivars carrying major resistance (R) genes has been the most effective approach for BB management. However, because of specific interaction of each R gene with the product of the corresponding pathogen avirulence or effector gene, new pathogen strains that can overcome the deployed resistance often emerge rapidly. To deal with ever-evolving Xoo, it is necessary to identify novel R genes and resistance quantitative trait loci (QTL).Bacterial blight (BB), caused by Xoo strains, namely Z173 (C4), GD1358 (C5), V from China and PXO339 (P9a) from Philippines. Using the 3 K RG 4.8mio filtered SNP Dataset, a total of 11 QTL associated with BB resistance on chromosomes 4, 5, 11 and 12 were identified through a genome-wide association study (GWAS). Among them, eight resistance loci, which were narrowed down to relatively small genomic intervals, coincided with previously reported QTL or R genes, e.g. xa5, xa25, xa44(t). The other three QTL were putative novel loci associated with BB resistance. Linear regression analysis showed a dependence of BB lesion length on the number of favorable alleles, suggesting that pyramiding QTL using marker-assisted selection would be an effective approach for improving resistance. In addition, the Hap2 allele of LOC_Os11g46250 underlying qC5–11.1 was validated as positively regulating resistance against strain C5.BB resistance of a diverse panel of 340 accessions from the 3000 Rice Genomes Project (3 K RGP) was evaluated by artificial inoculation with four representative Our findings provide valuable information for the genetic improvement of BB resistance and application of germplasm resources in rice breeding programs.The online version contains supplementary material available at 10.1186/s12284-021-00462-3. Oryza sativa L.), an important cereal crop that supports more than 50% of the world population, suffers many biotic and abiotic stresses that lead to serious damage to crop yield and food safety (Oerke Xanthomonas oryzae pv. oryzae (Xoo), is one of the most destructive bacterial diseases worldwide, in both tropical and temperate regions  genes and quantitative trait loci (QTL) that will support the development of new resistant varieties.Rice (ty Oerke . Rice baions Mew . As it iions Mew . Breedinions Mew . BecauseXa1 – xa-45(t)) that confer resistance to BB have been identified, and are distributed on all chromosomes except chromosomes 9 and 10  studies except for Xa43(t), which was discovered in a multi-parent advanced generation inter-cross (MAGIC) population through genome-wide association study (GWAS) ) have been cloned and characterized (https://shigen.nig.ac.jp/rice/oryzabase/). Because of either their lower level of resistance or their narrow spectrum of resistance, only a few genes  with broad-spectrum resistance have been widely deployed in breeding programs so far  was identified from a japonica MAGIC population of 120 lines and confirmed using a bi-parental population , PXO99 (P6), PXO339 (P9a) were identified in 172 indica rice accessions through GWAS, but no significant SNP for Chinese Xoo strains GD1358 (C5) or V was detected  , 65 GJ (Geng/Japonica), 30 cA (circum-Aus), 8 cB (circum-Basmati) and 11 Admix (accessions between XI and GJ groups) , GD1358 (C5), and V from China were used to evaluate the resistance of the 340 rice accessions. The representative strain of Philippine race 9 PXO339 (P9a), which was used in different panels for GWAS in previous studies . Using the linkage disequilibrium (LD) pruning tool of PLINK 1.9 .A total of 4,130,496 SNPs from 340 rice accessions with the criteria of genotype missing rate ≤ 5%, minor allele frequency ≥ 1%, were obtained for association analysis. The Balding–Nichols method was used to develop a kinship matrix  was considered as the local LD interval .According to the previous studies, a region containing more than two SNPs above the significant Haplotype analysis for candidate genes within each QTL was performed based on the significant SNPs and these genes can be classified into several major haplotypes (at least containing 10 accessions). Then combining with phenotype data, variations in LL explained by different haplotypes were analyzed by ANOVA. The genes that displayed significant LL differences among haplotypes were chosen for further research.Each SNP has two alleles differing by a single nucleotide variation. Alleles with negative effects leading to shorter LL (associated with a more resistant phenotype) are referred to as favorable alleles  with the resistant allele (Hap2) of LOC_Os11g46250. The amplification reaction was performed under the following condition: 95 °C 3 min; 95 °C 15 s, 60 °C 15 s, 72 °C 6 min, 35 cycles; 72 °C 5 min. The fragment was cloned into plant binary vector pCAMBIA1300  digested by BamH I and Hind III to generate a complementary construct with the native promoter. Then the complementary construct was introduced into Agrobacterium tumefaciens strain EHA105 and subsequently transferred to rice variety Nipponbare  following the manufacturer’s protocol via an ABI Prism 7900 Sequence Detection System . The rice ubiquitin gene (LOC_Os03g13170) was used as the internal control. All primers used in this study are listed in Table SThe genomic DNA fragment of 2 lines and wild-type Nipponbare were planted in the transgenic experiment base (the same growing condition with the experiment farm) at the Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing, China and inoculated with C5.To evaluate their resistance to strain C5, all the TBased on 527,165 independent SNPs with genotype missing rate ≤ 5% and minor allele frequency ≥ 1%, a phylogenetic tree was developed using the neighbor-joining method, dividing 340 accessions into four distinct major clusters , namely XI, GJ, cA and cB subgroups  showed large phenotypic variation  in 340 accessions inoculated with four on Fig. S. Among ton Fig. S. AnotherXoo strains were divided into two obvious groups, namely Philippines strain group (P9a) and Chinese strain group  . Also, for both strains V and P9a, the average LL of subgroup XI was significantly longer than that of subgroup cA (P < 0.05)  Fig. a. At the V) Fig. a. Accord V) Fig. b. For al05) Fig. c. These Xoo strains and one Philippines Xoo strain, we performed GWAS with a mixed linear model of the EMMAX program using 4,130,496 high quality SNPs and LL as genotype and phenotype data, respectively. Based on the effective number of independent markers, the threshold of significant P-value was estimated to be 6.31E− 8 by the Bonferroni correction method. In total, we identified 11 QTL within 1576 unique SNPs associated with BB resistance to four strains , which played an indispensable role in pollen and anther development, including anther wall programmed cell death , and 12. Notably, a cluster of significant SNPs on chromosome 11 were located in the genomic region 27,985,691–28,479,727 bp. Based on linkage disequilibrium analysis, the continuous region of chromosome 11 were divided into two QTL, namely qV-5.1 region harbored 272 significant SNPs, overlapping with a broadly effective recessive resistance gene xa5. Notably, no QTL or gene for BB resistance was previously reported in the other QTL, qV-5.2 , spanning about 31 kb on chromosome 5. This region contained 98 significant SNPs and might include potential novel BB genes.For strain V, there was a steep peak of resistance-related SNPs on chromosome 5, defined by two QTL. The qP9a-12.1–qP9a-12.3). Among them, qP9a-12.2 was located close to the reported race-specific resistance gene xa25.For strain P9a, we identified 1173 significant SNPs associated with BB resistance on chromosome 12. Based on linkage disequilibrium analysis, this region was divided into three QTL , while qC5–11.2 spanned about 49 kb. Of particular note, a recessive gene xa44(t), conferring resistance to Xoo isolate HB1009 (K3a) from Korea, was exactly located in the interval of qC5–11.1 (Kim xa44(t), Os11g0690466 and Os11g0690066. Using the 3 K RG 4.8mio filtered SNP Dataset, we found no SNP variation for Os11g0690466 among 340 accessions, while 120 SNPs for Os11g0690066 clustered the accessions into three major haplotypes  in the resistant and susceptible cultivars, and found no difference between them, suggesting that the SNP variation and expression of xa44(t) were not responsible for qC5–11.1 and other genes responsible for qC5–11.1 should be studied.The above GWAS results indicated chromosomes 5, 11, and 12 as hotspots for BB resistance. Strikingly, chromosome 11 contained multiple resistance loci for different 11.1 Kim , but no qC5–5 and qV-5.1 overlapped with LOC_Os05g01710 (xa5), a recessive gene encoding transcription initiation factor IIA subunit 2, responsible for broad-spectrum resistance  was located in the vicinity of transposon gene LOC_Os05g01770 , potentially the causal gene conferring resistance to C5. Three QTL for resistance to P9a were all located in the interval of L12 loci, in the range of 16,502,066–17,531,046 bp on chromosome 12, and were also reported to be associated with resistance to P9a  were not responsible for qC5–11.1, we focused on other genes in this region. In the candidate region of qC5–11.1 spanning 27,985,691–28,013,096 bp, two significant SNPs with P-value of 8.80E− 09 (rs11_28009272 and rs11_28011447) were exactly located within LOC_Os11g46250, which encodes an uncharacterized protein  is a representative strain of pathotype 4 from the Yangtze River basin  was also located in the region of qC5–11.1  were newly discovered in the present study, located a long distance on the physical map from known BB resistance QTL or genes , which contained 28 cis-regulatory elements  that are involved in pathogen, elicitors and disease resistance response , Xa30(t), Xa32(t), Xa35(t), Xa36(t), Xa39, Xa40, xa41(t), Xa43(t) and xa44(t), and cloned genes Xa3/Xa26, Xa4, Xa10, Xa21, Xa23 (https://shigen.nig.ac.jp/rice/oryzabase/). Because of the wide deployment of single R genes and Xoo-rice coevolution, elite R genes Xa4 and Xa21 have been overcome by the newly-emerged Xoo strains C5 and V in China  showed greater resistance to strains C4, C5, V and P9a than those carrying Hap1 , OS198 (C6) and IV than IR24, the recurrent parent of IRBB5 (data unpublished), indicating that xa5 conferred pleiotropic resistance to Xoo strains. As most accessions contain the susceptible allele of xa5, it is likely to improve BB resistance by editing this gene using the CRISPR/Cas9 technique , Os11g0690066Additional file 5: Fig. S1. Distribution of bacterial blight lesion length among 340 accessions. Box edges represent the 0.25 and 0.75 quantiles with median values indicated by bold lines. Fig. S2. Lesion length of accessions carrying different haplotypes of one candidate gene of xa44(t), Os11g0690066. Characters above boxplots indicate significant differences according to Duncan’s multiple comparison tests (P < 0.05). Fig. S3. Haplotype analysis of xa5. (A) Exon-intron structure and haplotypes based on significant SNPs. Rectangles and lines represent exons and introns, respectively and the coding sequence highlighted in blue. (B) Lesion length of accessions with different haplotypes. Box edges represent the 0.25 and 0.75 quantiles with median values indicated by bold lines. Characters above boxplots indicate significant differences according to Duncan’s multiple comparison tests (P < 0.05). Fig. S4. Haplotype analysis of xa25. (A) Exon-intron structure and haplotypes based on significant SNPs. Rectangles and lines represent exons and introns, respectively and the coding sequence highlighted in blue. (B) Lesion length of accessions with different haplotypes. Box edges represent the 0.25 and 0.75 quantiles with median values indicated by bold lines. Characters above boxplots indicate significant differences according to Duncan’s multiple comparison tests (P < 0.05). Fig. S5. Linear regression analysis of the number of favorable alleles and lesion length of Xoo strains (A) C5, (B) V and (C) P9a. Fig. S6. Relative expression level of Hap1 (A) and Hap2 (B) of LOC_Os11g46250 in transgenic lines and wild-type. UBQ denotes the rice ubiquitin gene (LOC_Os03g13170) as the internal control; ** refers to significant differences at P < 0.01; WT and CP-1/CP-2/CP-3 refer to wild-type Nipponbare and three independent transgenic lines carrying complementary genomic fragments, respectively. Fig. S7. Haplotype analysis of LOC_Os05g01760 underlying qV-5.2. (A) Exon-intron structure and haplotypes based on significant SNPs. Rectangles and lines represent exons and introns, respectively and the coding sequence highlighted in blue. (B) Lesion length of accessions with different haplotypes. Box edges represent the 0.25 and 0.75 quantiles with median values indicated by bold lines. Characters above boxplots indicate significant differences according to Duncan’s multiple comparison tests (P < 0.05).


MyoD) and myosin heavy chain-I (MyHC-I) transcripts, and the phosphorylation levels of protein kinase B (AKT), mammalian target of rapamycin (mTORC1), initiation factor 4E-binding protein 1 (4EBP1) and S6 kinase 1 (S6K1) in LT, while decreasing the expression of muscle atrophy F-box (MAFbx) and forkhead Box O1 (Foxo1) mRNA transcripts. Our results indicated that fermented diet improved meat quality and enhanced LT growth of finishing pigs by increasing insulin/AKT/mTORC1 protein synthesis cascade and activating Foxo1/MAFbx pathway, along with the regulation of ribosomal protein and proteins involved in muscle contraction and muscle hypertrophy. Little is known about the effect of fermented feed on lean mass production of finishing pigs and the underlying mechanism. This study aimed to investigate the effect of fermented diet on growth performance, carcass traits, meat quality and growth of longissimus thoracis (LT) of finishing pigs. Our results found that fermented diet significantly increased loin eye area and lean mass percentage, decreased backfat thickness and improved meat quality of LT by decreasing shear force and drip loss at 48 h post slaughter and improving meat sensory characteristics compared with control diet. Fermented diet also increased the abundance of insulin, insulin receptor, myoblast determination protein (IR), myoblast determination protein (MyoD) and myosin heavy chain-I (MyHC-I) transcripts, and the phosphorylation levels of AKT, mTORC1, 4EBP1 and S6K1 in LT, while decreasing the expression of muscle atrophy F-box (MAFbx) and forkhead Box O1 (Foxo1) mRNA transcripts. Moreover, proteomic analysis revealed that differentially expressed proteins predominantly involved in protein synthesis and muscle development were modulated by fermented diet. Our results indicated that a fermented diet improved meat quality and enhanced LT growth of finishing pigs by increasing insulin/AKT/mTORC1 protein synthesis cascade and activating the Foxo1/MAFbx pathway, along with the regulation of ribosomal protein and proteins involved in muscle contraction and muscle hypertrophy.This study investigated the effect of fermented diet on growth performance, carcass traits, meat quality and growth of longissimus thoracis (LT) of finishing pigs. A total of 48 finishing pigs  weighing 98.76 ± 1.27 kg were randomly assigned to two treatments (eight pens per treatment and three pigs per pen) for a 28-d feeding trial, including control diet and fermented diet. Fermented diet significantly increased the loin eye area and lean mass percentage, decreased backfat thickness and improved meat quality of LT by decreasing the shear force and drip loss at 48 h post slaughter and improving meat sensory characteristics compared with control diet. A fermented diet also significantly increased the abundance of insulin, insulin receptor ( Fermented feed, which was fermentation of plant materials with beneficial bacterial, has been extensively studied in attempts to improve feed utilization and decrease use of in-feed antibiotics . MicrobiThe effect of fermented feed on growth performance, gut microbiota and immunity of piglets has been widely reported ,13. HoweSeveral studies have investigated the effect of fermented feed on meat quality of finishing pigs and focused on lightness, redness, shear force, flavor and drip loss ,18,19. HThus, the present study aimed to investigate the effect of fermented feed on growth performance, meat quality and muscle growth in pig during the late-finishing phase. The underlying mechanism of skeletal muscle growth of LT modulated by fermented feed was also explored.All animal procedures used in this study were approved by the Animal Care and Use Committee of Guangdong Academy of Agricultural Sciences (authorization number GAASIAS-2016-017). All efforts were made to minimize any suffering of animals in accordance with the Chinese Guidelines for Animal Welfare of Science and Technology Ministry of China .8 cfu/mL), Bacillus subtilis (2 × 107 cfu/mL) and Lactobacillus reuteri (1.8 × 108 cfu/mL). The mixture was then transferred to a polythene bag (20 kg in capacity) equipped with a one-way valve at room temperature (25–35 °C) for at least 96 h. After fermentation, wet fermented samples (approximately 800 g) were collected for chemical analysis; the remaining fermented feed with a moisture content of 38 ± 2.5% was kept in a room at ambient temperature (16 °C). The basal diet (control diet) was formulated to meet the nutrient recommendations of the National Research Council 2012  . Both cog for 15 min. The supernatant was collected for pH and lactic acid analysis. pH was measured using an electronic pH meter . The lactic acid content was determined using a lactic acid assay kit in accordance with the protocol of the manufacturer . For chemical composition analysis, samples of control and fermented feeds were dried at 65 °C for 24 h and then ground using a hammer mill and sifted through a 1 mm screen for dry matter, crude fiber, neutral detergent fiber, acid detergent fiber, crude fat, starch, crude protein contents and amino acids analysis. Crude fiber, neutral detergent fiber, acid detergent fiber, crude fat, starch and crude protein contents of the sample were measured by the methods of the Association of Official Analytical Chemists  and calculated on an 88% dry matter basis. The amino acid profiles of feed samples were determined using an AA analyzer  by the post-column derivatization of ninhydrin. Before analysis, 0.1 g sample was hydrolysis with 10 mL 6 M HCl for 24 h at 115 °C. Hydrolyzates were then transferred to a 50 mL volumetric flask and diluted with 0.02 mol/L HCl. 1 mL diluent was transferred to a 6 cm glass dish and evaporated to dryness in a water bath at 65 °C. After dissolving with 2 mL HCl (0.02 mol/L) and filtering (0.22 μm), amino acids were fractionated on a protein hydrolysate analysis column  at 0.40 mL/min outflow rate. The individual amino acid was identified using standards  and calculated on an 88% dry matter basis. Both the fermented sample and basal diet (3 g) were suspended in 10 mL ultrapure water and centrifuged at 5000× A total of 48 finishing pigs  weighing 98.76 ± 1.27 kg were randomly assigned to two treatments (eight pens per treatment and three pigs per pen). The pigs were provided with a control diet or fermented diet entirely substitute for control diet. All pigs were housed in 16 adjacent pens (1.8 × 4.6 m) equipped with slatted floors in an environmentally-controlled facility. The pigs were fed three times a day  with the prepared diet in feeding troughs and had ad libitum access to feed and water. Feed leftovers in each pen were collected and quantified every day. The amount of feed consumed per pen was recorded every day to determine the average daily feed intake (ADFI) on an 88% dry matter basis. All pigs were weighed individually at the beginning and the termination of the experiment to calculate the average daily gain (ADG). The feed to gain ratio (F/G) were calculated from the feed intake (on an 88% dry matter basis) and the increased body weight during the whole experimental period.At the end of 39 d treatment (by when the body weight (BW) of control pigs were about 130 kg, 165 ± 5-d-old), one pig from each pen with medium BW in both control and experimental group was selected and transferred to the slaughterhouse within the Institute of Animal Science, Guangdong Academy of Agricultural Sciences. The pigs were electrically stunned, exsanguinated, eviscerated and divided lengthwise. The backfat depths over the first, 10th and last ribs were measured using a vernier caliper . Loin eye area was traced at the interface of 10th to 11th rib and measured by planimetry. Percentage of lean mass was estimated according to the guidelines of National Research Council (NRC) 1998: lean mass (%) = 4.40 + 0.54 × carcass weight (kg) − 4.21 × average back fat thickness (cm) + 0.18 × loin eye area.LT muscle samples  were removed for meat quality traits analysis. A chop (25 mm thick slice) for meat color and pH measurements was excised from LT sample between the 10th and 11th ribs. Meat color values  were measured at 45 min, 24 h and 48 h after slaughter using a Minolta CR-400 Chroma meter  with an 8 mm measuring port, D65 illuminant and a 10° observer angle, and the values of L *, a * and b * of each sample were recorded in triplicate. The pH of LT samples at 45 min, 24 h and 48 h post-mortem were determined using a pH meter , which was calibrated with pH 4.6 and 7.0 buffers before measurements.A chop (25 mm thick slice) was excised for shear force (N) analysis from the LT sample between the 11th and 12th ribs, vacuum packed and stored at 4 °C. At 24 h after slaughter, the muscle samples were placed in a plastic bag and cooked in a 80 °C water bath until the internal temperature reached 70 °C. Internal temperature was monitored during cooking with a hand-held temperature probe placed into the geometric center of each sample. Then cooked samples were cooled in running water to room temperature and were sheared parallel to the muscle fiber direction and cut into shaped strips (1 cm × 1 cm × 3 cm). Finally, the shear force (N) of the LT was measured using a digital-display-muscle tenderness meter  with a load cell of 15 kg and a 200 mm/min crosshead speed. Fifteen replicates of each sample were recorded. A chop (30 mm thick slice) was excised for drip loss analysis from the LT sample between the 13th and 14th ribs. Drip loss of LT samples were determined on weighed cubes of muscle (about 30 g), suspended on hooks in airtight plastic bags, stored at 4 °C for 24 h and 48 h and reweighed after removing surface moisture. Drip loss was calculated as:Intramuscular fat content was determined using Soxhlet extraction . ApproxiA chop (25 mm thick slice) that was excised for sensory evaluation from the LT sample between the 12th and 13th ribs was vacuum packed and stored in a refrigerator (4 °C). At 4 h after slaughter, meat samples were taken out, trimmed and cut into eight cubes of about 1.8 cm dimension. Each sample cube was placed in a bowl, sealed with aluminum foil and labeled with a random code. Bowls containing samples were cooked at 120 °C in a humid oven for 10 min then cooled in a water bath (50 °C) until served to eight panelists, who have experience in sensory assessment, across one session. There was about 4 min for panelists to evaluate each sample and each panelist was asked to test 16 samples for seven subjective traits, including tenderness, juiciness, flavor, fragrance, off-flavors, broth freshness and color. Distilled water (about 30 °C) was provided for panelists to cleanse their palate between samples. Sensory variables were rated using eight-point scales: tenderness , juiciness , flavor , fragrance , off-flavors , broth freshness  and color .LT sample between the 10th and 11th ribs was immediately collected and frozen in liquid nitrogen, and then stored at −80 °C for qPCR. Trizol reagent  was used to isolate total RNA from LT sample, according to the manufacturer’s instructions. Purity and concentrations of RNA were assessed using a NanoDrop-ND1000 spectrophotometer . Total RNA (1 μg) was used to synthesize cDNA using a PrimeScript™ II 1st Strand cDNA Synthesis Kit . SYBR green I , 10-fold diluted cDNA and gene-specific primers  in a fing for 15 min at 4 °C). Concentrations of extracted protein were measured by a BCA protein assay kit  with bovine serum albumin (BSA) standards. Equal quantities of protein were diluted with 5× loading buffer, denatured at 100 °C for 10 min, cooled on ice and then used for Western blots. Denatured proteins were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and then, separated proteins were transferred to nitrocellulose membranes. After blocking with 3% BSA in tris buffered saline tween (TBST) buffer for 10 min, membranes were washed three times, then incubated with the diluted primary antibodies overnight at 4 °C. Subsequently, after washing three times, membranes were then incubated with the appropriate HRP-labeled secondary antibodies for 1 h at room temperature. After three washes, immunoreactive proteins were visualized with chemiluminescent HRP substrate  and a VersaDoc imaging system . Image J software  was used to quantify the band of protein intensity. Primary antibodies for β-actin (Cat number: #4970), p-AKT (Cat number: #4060), p-mTOR (Cat number: #2971S), p-4EBP1 (Cat number: #62855S) and p-S6K1 (Cat number: #2215S) were all from Cell Signaling Technology, Boston, MA). The results are expressed as the abundance of each target protein relative to β-actin.An LT sample between the 10th and 11th ribs was immediately collected and frozen in liquid nitrogen, and then stored at −80 °C for western blot analysis. About 0.5 g frozen sample was lysed for 30 min at 4 °C in 1 mL of RIPA buffer containing 1% protease inhibitor cocktail and 1% phosphatase inhibitor. The sample lysates were centrifuged  procedure described by Wisniewski et al., and the resulting peptide mixture was labeled using the 4-plex/8-plex iTRAQ reagent according to the manufacturer’s recommendations  . BrieflyFor labeling, each iTRAQ reagent in 70 μL ethanol was added to the respective peptide mixture. The samples were labeled as (Sample 1)-114, (Sample 2)-115, (Sample 3)-116 and (Sample 4)-117, and then multiplexed and vacuum dried. 2PO4 in 25% of ACN, pH 2.7), and then loaded into a 4.6 × 100 mm Polysulfoethyl column . Then, the peptides were eluted at a flow rate of 1 mL/min with a gradient of 0–10% buffer B  for 2 min, 10–20% buffer B for 25 min, 20–45% buffer B for 5 min and 50–100% buffer B for 5 min, respectively. Elution procedure was monitored by absorbance at 214 nm, and fractions were collected every 1 min. The collected fractions (about 30 fractions) were finally combined into 10 pools and desalted on C18 Cartridges (Empore™ SPE Cartridges C18 (standard density), bed I.D. 7 mm, volume 3 mL, Sigma). Each fraction was concentrated by vacuum centrifugation and then reconstituted in 40 µl of 0.1% (v/v) trifluoroacetic acid. All samples were stored at −80 °C and used for LC-MS/MS analysis.iTRAQ labeled peptides were fractionated by Strong Cation Exchange (SCX) chromatography using the AKTA Purifier system . The dried peptide mixture was reconstituted and acidified with 2 mL buffer A (10 mM KHm/z) for high energy collisional dissociation (HCD) fragmentation. Determination of the target value is based on predictive Automatic Gain Control (pAGC). Dynamic exclusion duration was 60 s. Survey scans were acquired at a resolution of 70,000 at m/z 200 and resolution for HCD spectra was set to 17,500 at m/z 200. Normalized collision energy was 30 eV and the underfill ratio, which specifies the minimum percentage of the target value likely to be reached at maximum fill time, was defined as 0.1%. The instrument was run with peptide recognition mode enabled.Liquid Chromatography (LC)-Electrospray Ionization (ESI) Tandem MS (MS/MS) was analyzed by Q Exactive. Experiments were performed on a Q Exactive mass spectrometer that was coupled to Easy nLC . Ten μL of each fraction was used for nano LC-MS/MS analysis. The peptide mixture (5 μg) was loaded onto a C18-reversed phase column  in buffer A (0.1% Formic acid) and separated with a linear gradient of buffer B (80% acetonitrile and 0.1% Formic acid) at a flow rate of 250 nl/min handled by IntelliFlow technology exceed 140 min. MS data were obtained using a data-dependent top 10 method, dynamically choosing the most abundant precursor ions from the survey scan .The MS/MS spectra were searched against the uniport Sus scrofa database for peptide identification and quantification using Mascot 2.2  and Proteome Discoverer1.3 . The following options were used to identified protein: Peptide mass tolerance = 20 ppm, MS/MS tolerance = 0.1 Da, Enzyme = Trypsin, Missed cleavage = 2, Fixed modification: Carbamidomethyl (C), iTRAQ4/8plex(K), iTRAQ4/8plex(N-term), Variable modification: Oxidation(M), false discovery rate (FDR) ≤ 0.01. Additionally, fold change of ≥1.20 or ≤0.83, p < 0.05. Growth performance was analyzed using the following model: y = μ + α + β, where y is the response variable, μ is the overall mean, α is the fixed effect of diet and β is the random effect of pen. Carcass traits and sensory evaluation analysis were analyzed using y = μ + α + β model, where y is the response variable, μ is the overall mean, α is the fixed effect of diet and β is the random effect of pig identification, panelist number and tasting sample order. The other traits were analyzed using GraphPad Prism Version 5  and presented as means ± SEM. Significance was evaluated using a two-tailed unpaired t test. p < 0.05was considered to be a statistically significant difference.The data of growth performance, carcass traits and sensory evaluation of finishing pigs were analyzed using SAS software , and presented as means ± SEM. Statistical significance was considered at As shown in p < 0.1). The fermented diet significantly decreased the last rib fat depth (p < 0.01) while increasing the loin eye area and lean mass percentage compared with the control diet (p < 0.01). The meat quality indices of LT are summarized in p < 0.05) and shear force (p < 0.01) compared to that from pigs fed the control diet. Additionally, the fermented diet significantly increased the scores for tenderness, fragrance and broth freshness (p < 0.05) but had no effect on the other sensory parameters including juiciness, flavor off-flavors and color of LT (As shown in or of LT . p < 0.05), IR (p < 0.01) and MyoD (p < 0.01), and the same trend for the expression of myogenic factor 5 (MYF5) and cyclinD1 mRNA, although there were no significant statistics, while decreasing the abundance of MAFbx and Foxo1 transcripts (both p < 0.05). The fermented diet had no effect on the expression of insulin like growth factor 1 (IGF-1), insulin-like growth factor 1 receptor (IGF-1R), myogenin (MyoG), muscle ring finger 1(MURF1) and forkhead box O4 (Foxo4) compared with the control diet. As for the MyHC genes expression levels, the results showed that the fermented diet significantly increased the abundance of MyHC-I, and had no effect on fast-oxidative type IIa (MyHC-IIa), fast-oxidative glycolytic type IIx (MyHC-IIx) and fastglycolytic IIb (MyHC-IIb) (p < 0.05), mammalian target of rapamycin (mTOR) (p < 0.05), S6 kinase 1(s6k1) (p < 0.01) and initiation factor 4E-binding protein 1(4EBP1) (p < 0.05) compared with the control diet (yHC-IIb) B. At therol diet C,D.The analysis identified 1549 proteins in LT, and 124 of them were DEPs between the two diets. Compared with the control diet, 80 DEPs were increased while 44 DEPs were decreased by the fermented diet A. VolcanMyoD, CSRP3 and FAU, while it was negatively related to the abundance of MAFbx.A Pearson correlation analysis was used to investigate the associations between genes and proteins expression and lean percentage . The resIn the present study, we found that microbial fermentation decreased crude fiber and pH value, and increased the lactic acid concentration, crude protein content and amino acid profiles of the feed. The increased crude protein after fermentation was consistent with previous fermented soybean meal (SBM) ,25, fermMyHC-I, which contributes to increase water holding capacity and tenderness [Several studies have reported that fermented feed had no significant effect on ADFI and ADG of finishing pigs ,17. Consnderness , furtherIn the present study, the fermented diet significantly increased loin eye area and lean mass percentage. A study by Ahmed et al. showed that fermented herb diet increased lean production , but theFoxo1 and MAFbx. Additionally, a previous study reported that leucine also increased the mRNA and protein levels of MyoG and MyoD [MyoD by the fermented feed that was rich in amino acid profile. Consequently, the proteolysis-related FOXO1/MAFbx pathway and protein synthesis-related MyoD pathway may serve as a target for muscle metabolism and growth when subjected to increased amino acid profile of the feed after fermentation.The modulation of skeletal muscle mass depends on the balance between protein synthesis and degradation. Previous studies have shown a reduction in protein degradation after branched-chain amino acids or leucine administration through changing the expression of Foxo1, MAFbx and MuRF-1 ,43,44,45and MyoD , which wand MyoD . SimilarOn the other hand, ribosomal proteins and muscle contraction-related proteins have been shown to play an important role in protein synthesis during myogenesis and promote muscle growth ,49,50,51In summary, the promotion of muscle growth by the increased amino acid profile of the feed after fermentation was regulated, at least in part, by increasing the insulin/AKT/mTORC1 protein synthesis cascade and activating Foxo1/MAFbx pathway, along with the regulation of ribosomal protein and proteins involved in muscle contraction and muscle hypertrophy. Additionally, a fermented diet improved meat quality of longissimus thoracis (LT) by decreasing the shear force and drip loss at 48 h post slaughter and increasing sensory characteristics without affecting the IMF content.


Matricaria chamomilla is a well-documented medicinal herb that possesses anti-inflammatory, antioxidant and antitumor activities and is commonly used to treat diverse ailments. The present study aimed to reveal the hepatoprotective effects of Matricaria chamomilla aqueous extract during an intermediate stage of colorectal cancer (CRC) in mice. Male Balb/c mice were divided into six groups: group A served as control, group B received chamomile extract (150 mg/Kg b.w.) orally for 12 weeks, and groups C-F received weekly intraperitoneal injections of DMH (20 mg/Kg b.w.) once a week for 12 weeks. In addition to DMH, groups D and F received chamomile during the initiation and post-initiation stages, respectively. Blood and liver samples were collected for biochemical and molecular analyses. The results showed that DMH induced hepatic injury in mice as shown by significant increase in serum aspartate aminotransferase and alanine aminotransferase. The changes in biochemical parameters were accompanied by activation of the Wnt signaling pathway leading to increased hepatocytes proliferation as well as inflammation evidenced by high levels of pro-inflammatory enzymes cyclooxygenase 2 (COX-2) and inducible nitric oxide synthase (iNOS). The results also showed potential hepatoprotective effects of chamomile extract against DMH-induced liver injury, proliferation and inflammation. Chamomile restored the biochemical and molecular parameters and this improvement was more pronounced in mice pretreated with the extract. In conclusion, chamomile extract may exert its hepatoprotective activities against DMH probably due to the antioxidant, antiproliferative and anti-inflammatory properties of its flavonoids.Dimethylhydrazine (DMH) is a potent colonic and hepatic carcinogen that is metabolized into oxyradicals causing liver injury and DNA mutations.  Dimethylhydrazine; Chamomile; liver injury; Wnt signaling; COX-2; iNOS; Biochemistry; Biological Sciences; Cancer Research; Hepatobiliary System; Molecular Biology; Oncology; Pharmaceutical Science; Microbiology; Pharmaceutical Chemistry; Toxicology CRC dev1,2-Dimethylhydrazine (DMH) is a toxic environmental pollutant  that hasMatricaria chamomilla L., is one of the most commonly used medicinal herbs whose extracts and standardized tea are usually prepared from the dried flowers [Asteraceae) that has been traditionally used in treating wounds, eczema, ulcers, gout, skin irritations, burns, neuralgia, rheumatic pain, hemorrhoids, diaper rash, chicken pox, ear and eye infections, and respiratory disorders [Since conventional and synthetic drugs used in the treatment of diseases, including cancer, have a vast array of unfavorable side effects, there is an increasing worldwide interest in the use of traditional medicinal herbs to treat various diseases . The the flowers . Chamomiisorders ,22. In aisorders , 24, 25.Chamomile contains different bioactive constituents such as the blue oil (0.24%–1.9%) containing terpenoids, α-bisabolol and chamazulene, farnesene, spiro-ether quiterpene lactones, hydroxycoumarins, glycosides, flavanoids , coumarins (herniarin and umbelliferone), and terpenoids . Chamomiet al. [Recently, a study by El Joumaa et al.  revealedet al. , we hypo22.11,2-Dimethylhydrazine dihydrochloride was obtained from ACROS Organics™ . Phenylmethanesulfonylchloride (PMSF) was purchased from Roche Diagnostics . All primers were purchased from BIO-RAD®  except GAPDH primers which were synthesized by TIB Molbiol . All other chemicals and reagents used were of high commercial and analytical grades.2.2Air-dried chamomile flowers of Syrian origin were purchased from a local market in Saida city, Lebanon. The taxonomic identification of this herb was performed by Dr. Salwa Mahmoud Abdul Rahman, Department of Biological Science, Faculty of Science at Beirut Arab University. Chamomile's flowers (2.5 g) were soaked in 100 mL of boiled distilled water (100 °C) and steeped at room temperature for 30 min with occasional stirring. The mixture was then filtered, aliquoted and stored at -20 °C to be used.2.3The aqueous extract was filtered with 0.25 μm Millipore SPE cartridges and diluted 1:10 with LCMS grade water. The resultant crude solution was injected into a UPLC-PDA  using a C18-Hypersil Gold reverse phase column to acquire a fingerprint 3D chromatogram. Gradient elution was performed with 0.1% formic acid in water/acetonitrile at a constant flow rate of 0.285 mL/min and an injection volume of 10 μL. Separation was carried out in 30 min.et al. [https://www.ncbi.nlm.nih.gov/pccompound).A list of 50 common polyphenols and flavonoids  was formet al.  and by P2.4ad libitum access to standard mouse diet and tap water. Mice were left to acclimate with these conditions for one week before beginning the experiments. Experimental procedures were carried according to the approved guidelines of the Institutional Review Board (IRB) at Beirut Arab University code number 2018A-0033-S-M-0245.Healthy 6-week-old male albino Balb/c mice were obtained from Beirut Arab University's animal facility. They were housed under standard laboratory conditions of light (12-hour light/dark cycle), temperature (22 ± 2 °C), and humidity with 2.5Animals were randomly divided into six experimental groups of 6 mice each. The experimental protocol is shown in The concentration used to induce CRC was based on Gurley, Moser & Kemp . The selAt the end of the treatment, mice were fasted overnight, then sacrificed and their livers were excised for further histological, biochemical and molecular analyses.2.6Liver tissues were homogenized in phosphate buffered saline  supplemented with 1 mM of the protease inhibitor PMSF at a ratio of 1 g per 5 mL of buffer. The homogenate was then centrifuged for 15 min at 15,000 rpm at 4 °C, and the supernatant was collected and stored at – 80 °C for later use.et al. using BSA  as a standard [Proteins concentration in all tissue homogenates were quantified according to the method of Lowry standard . Each sa2.7ALT and AST activities in all tissue homogenates were measured using the GPT (ALT) colorimetric assay kit  and GOT (AST) assay colorimetric kit , respectively and as recommended by the manufacturer.2.82.8.1Total RNA was extracted from liver homogenates using the RNeasy Plus Mini Kit  according to manufacturer recommendations. In brief, 200 μL of liver tissue homogenate were lyzed in 400 μL of denaturing guanidine-thiocyanate–containing RLT buffer, which inactivates RNases and ensures intact RNA isolation. The lysate was then passed through a gDNA eliminator spin column to eliminate genomic DNA. Ethanol was also added to the lysate in order to create conditions that promote selective binding of RNA to the RNeasy column. The sample was then applied to the RNeasy spin column, where total RNA binds to the membrane and contaminants are efficiently washed away. High-quality RNA was finally eluted in 80 μL RNase-free water.To check for the integrity of the eluted RNA, samples were electrophoretically separated on 1% agarose and visualized by UV illumination using ethidium bromide staining. RNAs appeared as two sharp bands corresponding to the 28S rRNA and 18 S rRNA. RNA was quantified through its absorbance which was measured at 260 nm. Its purity was assessed from the 260/280 absorbance ratio.2.8.2RNA was transcribed using the QuantiTect® Reverse Transcription Kit  according to manufacturer recommendations. In brief, 1.0 μg of RNA samples were incubated in 3 μL gDNA Wipeout Buffer and 9 μL of RNase free water at 42 °C for 2 min to effectively remove contaminating genomic DNA in a total volume of 14 μL. After genomic DNA elimination, RNA samples were reverse transcribed using Quantiscript Reverse Transcriptase (1.5 μL), Quantiscript RT Buffer (6 μL), and RT Primer Mix (1.5 μL) in a final volume of 20 μL. The reaction took place at 42 °C for 15 min and the enzyme was then inactivated at 95 °C for 3 min. Finally, the cDNA obtained were stored at –80 °C for later use.2.8.3The expression of Wnt signaling genes were quantified by RT-PCR using QuantiFast® SYBR® Green PCR Kit . The amplification reaction was carried out at final volume of 10 μL containing 5 μL of 2x QuantiFast SYBR Green PCR Master Mix, 1 μL (1 μM) of each primer (forward and reverse), 2 μL of cDNA and 1 μL of RNase-free water. Cycling was performed as follows. First, a denaturation step at 95 ° C for 5 min, followed by 45 cycles of denaturation at 95 ° C for 10 s and annealing/extension at 60 ° C for 30s.Forward (F) and reverse (R) sequences are shown in −ΔΔCt) where:Gene expression was measured by comparative threshold cycle (Ct) method using glyceraldehyde-3 phosphate dehydrogenase (GAPDH) as a reference gene. For each gene, the mean Ct (mCt) values were determined. ΔCt value was determined as the difference between the Ct of gene of interest and the Ct of GAPDH gene. The relative quantity of gene of interest expression compared to GAPDH gene was calculated applying the gene dosage ratio formula  according to manufacturer recommendations. The activity of iNOS was measured using Nitric Oxide Synthase Assay Kit  according to manufacturer recommendations.2.10P < 0.0001, ∗∗∗ at P < 0.001, ∗∗ at P < 0.01, and ∗ at P < 0.05.All statistical analyses were performed using Microsoft Excel, and they are shown as mean with standard deviations. Statistical significance was assessed using One-way ANOVA test followed by Tukey test. Graphs were drawn using GraphPad prism software and statistical significance was reported with a p-value < 0.05 considered as significant. Results with ∗∗∗∗ indicate the significance at 33.11  was obtained as compared to Group C. Likewise, chamomile post-treatment (Group F) significantly reduced the levels of AST and ALT by ~33% (P < 0.01) and 37% (P < 0.05), respectively, compared to the untreated mice in Group E.3.33.3.1P < 0.05) and β-catenin by 1.58 folds (P < 0.001) compared to the control group. Similarly, DMH-treated groups for 24 weeks (Group E) showed a significant upregulation in Wnt5a gene by 4.5 folds (P < 0.01) compared to the control group. However, chamomile pre-treatment of DMH-injected mice (Group D) significantly downregulated the expression level of Wnt5a  and β-catenin  genes, compared to those receiving the carcinogen only (Group C). Chamomile post-treatment of DMH-injected mice (Group F) significantly downregulated the expression level of β-catenin  compared to Group E.As shown in 3.3.2P < 0.0001) respectively, compared to the normal control (Group A). However, pre-treated mice with chamomile extract (Group D) significantly increased the expression level of GSK3β and APC gene by 5.85 and 4.66 folds (P < 0.0001) respectively, compared to Group C. Chamomile, as a post-treatment (Group F), exerted a significant upregulation in the expression levels of APC gene by 4.54 folds in the liver (P < 0.0001) compared to group E.In Panels C and D of 3.3.3P < 0.0001) respectively, compared to the control group as illustrated in Panels E and F of P < 0.0001) respectively.DMH administration for 12 weeks (Group C) and 24 weeks (Group E) induced significant upregulation in the expression of Tcf4 gene by 5 folds and 9.4 folds (P < 0.0001).As for the Lef1 gene expression, DMH administration for 12 weeks upregulated the level of Lef1 by 2 folds, and this effect was significantly reversed by chamomile pre-treatment which downregulated the expression level of this gene by 2.4 folds (3.4P < 0.0001) respectively, as well as the levels of Cyclin D1 gene by 2.9 (P < 0.01) and 6.2 folds (P < 0.0001) respectively, compared to the control. However, pre-treatment with chamomile extract caused a significant downregulation in the expression level of c-Myc and Cyclin D1 by 3.7 folds (P < 0.001) and 2.5 folds (P < 0.01) respectively, compared to group D. Similarly, chamomile as a post-treatment caused significant downregulation in the expression level of c-Myc gene by 8 folds and Cyclin D1 gene by 4.6 folds (P < 0.0001) respectively compared to group E.As shown in 3.5P < 0.05) after 12 and 24 weeks respectively, compared to the control group as shown in P < 0.05) respectively, compared to groups C and E. As for iNOS, chamomile pre-treatment of DMH-injected mice (Group D) led to a significant decrease in its activity by 56.7% (P < 0.05) compared to group C.DMH induced a significant elevation in COX-2 level by 40% and 40.7% (4M. chamomilla extract during an intermediate DMH-induced mouse model of CRC, taking into consideration the activation of Wnt signaling pathway and inflammation.CRC occurs as a result of many multistep processes that lead to the accumulation of mutations in tumor-suppressor genes and oncogenes . Recentlet al. [Matricaria chamomilla treatment in streptozotocin-induced diabetic rats [At the biochemical level, DMH-induced hepatotoxicity was evidenced by significant elevations in the levels of serum ALT and AST that are attributed to liver injury. The increments of serum ALT and AST activities are usually due to their leakage from liver cytosol into blood in cases of necrosis or membrane damage, making these enzyme potent markers of hepatic damage . The preet al.  that reptic rats . Likewistic rats , exertedtic rats , and redtic rats .At the molecular level, since DMH metabolites were shown to alkylate DNA causing mutations in Apc and β-catenin gene which are key players of the Wnt pathway , 14, 15,2O2 released from colonic tumors [2O2 into the circulation which can then reach the liver for detoxification [2O2 – by the DMH-induced colon tumors – that has been transferred to the liver for detoxification. Moreover, Burton et al. showed that cancer cells tend to protect themselves and grow by releasing products of lipid peroxidation which also explains the continuous liver damage for weeks after DMH exposure [It is quite clear from the obtained findings that a significant hepatic damage has been elicited, in the form of elevated AST and ALT levels and activated Wnt signaling pathway, after DMH treatment. While gene changes reflect long term damage attributed to DNA alkylating ability of DMH metabolites , chemicac tumors . A studyfication . Therefoexposure .M. chamomilla. The hepatoprotective and anti-proliferative effects of these flavonoids have been extensively studied in the literature. For example, apigenin has been shown to possess cancer-preventive and anti-cancer activities against different types of cancers via inhibiting the Wnt/β-catenin signaling [et al. showed that apigenin exerts anti-hepatoma activities via an apoptotic mechanism that is mediated through the p53-dependent pathway and the induction of p21 expression leading to cell cycle arrest in G2/M phase [The putative hepatoprotective and anti-proliferative activities of chamomile extract might be explained at least in part by the well-documented activities of its bioactive compounds. Phenolic compounds, such as flavonoids, tannins, coumarins, lignans, and quinones, are secondary compounds known for their hepatoprotective, antitumor and anti-inflammatory activities . In the ignaling ,54. In a/M phase . Luteoli/M phase . In CRC,/M phase . Also, l/M phase , and aga/M phase . Other p/M phase , 62, 63.et al. showed that luteolin – a major flavonoid found in chamomile – decreased the expressions of iNOS and COX-2 in AOM-induced CRC in mice [Moreover, cancers involve serious complications of inflammation, where the enzymes COX-2 and iNOS provide a link between inflammation and carcinogenesis . The Wnt in mice  and indu in mice .et al. [More importantly, our findings are in concomitance with a study by El Joumaa et al.  who showet al. [et al. [et al. [In comparison with other herbs and dietary agents, similar effects were observed in a study by Devasena et al.  where a  [et al.  and Jrah [et al.  showed t [et al. .M. chamomilla extract is a natural dietary agent with profound biological and pharmacological properties that ameliorate the hepatic damage induced by the carcinogen DMH. Further investigations are needed to provide assessments of oxidative stress and DNA damage caused by DMH. Also, our data open up future work for additivity and/or synergism of chamomile extract with Wnt/COX-2/iNOS inhibitors for the development of more potent therapies with minimal side effect, and identification of the active molecules responsible for antiproliferative and hepatoprotective activities.In conclusion, J. Borjac: Conceived and designed the experiments; Analyzed and interpreted the data; Wrote the paper.S. Shebbo and R. Kawash: Performed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data.M. El Joumaa: Performed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.The authors declare no conflict of interest.No additional information is available for this paper.