Extracellular cyclophilins (eCyps) A and B are chemotactic mediators in several illnesses in which inflammation plays an important role such as diabetes and cardiovascular diseases. Recently, eCypC has been reported as a potential biomarker for coronary artery disease but its effect in endothelium has not been determined. Moreover, there is a lack of studies with all these proteins in the same model, which makes difficult a direct comparison of their effects. In this work, MS1 pancreatic microendothelial cells were treated with eCyps A, B and C and their impact on endothelial function was analysed. eCyps A and C stimulated the release of IL-6 and MCP-1 and increased the expression of the receptor CD147, but eCypB did not affect these pro-inflammatory markers. Moreover, eCypC activated the translocation of NFkB-p65 to the nucleus. All these effects were reversed by pre-treatment with cyclosporine A. eCyps also produced endothelial dysfunction, as evidenced by the decrease in eNOS activation. Finally, the crosstalk among eCyps addition and their protein and gene expression was evaluated. eCypA generated a depletion in its protein and gene levels, whilst eCyps B and C upregulated their own protein expression. Moreover, each eCyp altered the intracellular expression of other Cyps, including cyclophilin D. This work is the first report of eCyps influence on iCyps expression, as well as the first description of eCypC as an activator of CD147 receptor and a mediator of endothelial dysfunction, which points to a potential role of this protein in vascular complications associated to diabetes. Diabetes-induced endothelial dysfunction is a critical factor in the initiation of micro- and macrovascular complications associated to this pathology. Along with the functional abnormalities found in large blood vessels, arterioles and capillaries are also affected by the damaging environment generated by chronic hyperglycaemia . Islets Extracellular cyclophilin A (eCypA) is an inflammatory mediator that contributes to endothelial malfunction by affecting the expression of adhesion molecules such as E-selectin, activating mitogen-activated protein kinases (MAPK) and reducing eNOS activity . This prcis to trans conformation and show affinity for the immunosuppressant drug cyclosporine A (CsA) (CypA belongs the cyclophilin (Cyp) family, composed by immunophilins with peptidyl-prolyl isomerase (PPIase) activity that catalyse the conversion of molecules from  A (CsA) . Cyps ar A (CsA) . It is s A (CsA) . CypC ha A (CsA) . Recent  A (CsA) . Moreove A (CsA) .via activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NFkB). The effects of eCypB are contradictory, since its binding to CD147 induces extracellular signal-regulated kinase (ERK) activation and migration of T-cells and monocytes (Extracellular CypA (eCypA) and extracellular CypB (eCypB) activities are mediated by their binding to the receptor CD147, a ubiquitous expressed type I transmembrane glycoprotein of the immunoglobulin superfamily found in neutrophils, lymphocytes, endothelial and vascular smooth cells . The intonocytes , but eCyonocytes . On the onocytes .Although the role of extracellular and intracellular Cyps A and B in inflammation-related diseases has been described and both proteins share a common receptor, the potential effect of eCyps on their own intracellular levels has not been explored before. In a previous study, we observed a correlation among serum levels of eCyps and the intracellular expression of these proteins in T-lymphocytes from coronary artery disease patients , which pIn this study, MS1 murine pancreatic microendothelial cells were treated with eCypA, eCypB and eCypC and their effects on endothelial dysfunction and CD147 expression were determined. Moreover, the crosstalk between elevated eCyps levels and their intracellular expression was evaluated. This is the first work that compares the effects of the three eCyps on the same cell line and describes the relationship among their extracellular and intracellular levels, as well as the first description of the potential role of eCypC on endothelial dysfunction.Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum, penicillin and streptomycin, 0.05% trypsin/EDTA, FITC-CD147 mouse monoclonal antibody, Pierce TM Protease Inhibitor Mini Tablets, phosphate buffered saline (PBS) (pH 7.2), Pierce TM Phosphatase Inhibitor Mini Tablets, Supersignal West Pico Luminiscent Substrate, Supersignal West Femto Maximum Sensitivity Substrate, oligo-dT primers, RevertAid Reverse Transcriptase and PowerUp SYBR Green Master Mix were purchased from Thermo Fisher Scientific . Recombinant human CypA protein (endotoxin level <1.0 Eu/µg), CsA, anti-cyclophilin F, anti-sodium/potassium ATPase, anti-NFkB-p65 and anti-lamin B1 antibodies were from Abcam . Recombinant human CypB (endotoxin level <1.0 Eu/µg) was from Antibodies-online and recombinant human CypC (endotoxin level <1.0 Eu/µg) was obtained from BioVendor . Mouse High Sensitivity T Cell Magnetic Bead Panel, PVDF membranes, anti-phospho-eNOS (S1177), anti-eNOS and anti-β-actin antibodies were obtained from Merck . Anti-CypA primary antibody and anti-CypB antibody were purchased from Elabscience . Anti-CypC antibody was obtained from Proteintech . Sodium dodecyl sulphate polyacrylamide gels and Bradford reagent were purchased from Biorad . Detachin™ solution was obtained from Genlantis . Another reagent grade chemicals were obtained from Sigma-Aldrich .2, containing a phosphatase/protease inhibitor cocktail. Nuclear extraction solution was composed by 100 mM Tris-HCl (pH 7.4), 2 mM Na3VO4, 100 mM NaCl, 1% Triton X-100, 1 mM EDTA, 10% glycerol, 1 mM EGTA, 0.1% SDS, 1 mM NaF, 0.5% deoxycholate, and 20 mM Na4P2O7, containing 1 mM PMSF and a protease inhibitor cocktail.Lysis buffer used for protein extraction of membranous and cytosolic fractions was composed by 50 mM Tris HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA and 1% Triton x-100, supplemented with a complete phosphatase/protease inhibitor cocktail. Hypotonic buffer used for nuclear and cytosolic lysates was composed by 20 mM Tris-HCl pH 7.4, 10 mM NaCl and 3 mM MgCl2 and 95% air and dissociated weekly using 0.05% trypsin/EDTA.MS1 murine pancreatic endothelial cell line was obtained from American Type Culture Collection (ATCC), number CRL2279. Cells were cultured in DMEM with high glucose (4.5 g/L) supplemented with 5% fetal bovine serum, 100 U/mL penicillin and 100 μg/ml streptomycin. Cells were maintained at 37°C in a humidified atmosphere of 5% COin vitro and in vivo rodent models provide valuable outcomes (Due to the great range of eCyps levels reported in patients (among 7.8 pg/ml and 3 μg/ml) , eCyps coutcomes . In this4 cells per well and allowed to growth for 24 h. Then, cells were treated with eCypA, eCypB and eCypC at 0.25 and 0.5 μg/ml for 24 h. LPS at 1 μg/ml was used as positive control. To confirm that cell activation was produced by eCyps, cells were pre-treated with 0.2 µM CsA for 1 h followed by addition of immunophilins. After incubation, cell medium was collected and kept at -80°C until analysis. Experiments were carried out by duplicate three independent times (n = 3). Levels of interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) were determined with a Mouse High Sensitivity T Cell Magnetic Bead Panel , following manufacturer’s instructions. Fluorescence was analyzed with Luminex 200™ instrument and xPONENT® software .MS1 cells were seeded in 96-well plates at 2 × 10n = 3).The expression of CD147 receptor was determined as previously described, with modifications . MS1 celMS1 cells were cultured in 12-well plates at 80% of confluence and allowed to settle down for 24 h. Cells were pre-treated with 0.2 µM CsA for 1 h and stimulated with eCypA, eCypB and eCypC at 0.5 μg/ml for 24 h.Membranous and cytosolic protein fractions were obtained as previously described . Cells wFor obtaining nuclear and cytosolic fractions, cells were washed with ice-cold PBS and 100 µL of hypotonic fraction were added. Cells were incubated for 15 min on ice and centrifuged at 3,000 rpm and 4°C for 15 min. The supernatant obtained was collected as the cytosolic fraction and the pellet was resuspended in the nuclear extraction solution. Samples were incubated on ice for 30 min with vortexing intervals of 10 min. Next, lysates were centrifuged at 14000 g and 4°C for 30 min and the supernatant was separated as the nuclear fraction. Cytosolic fraction was quantified with Direct Detect system (Merck Millipore) and nuclear protein concentration was determined by Bradford method.n = 3).All the assays were performed three independent times (® semi-dry transfer cell system was used for proteins transfer to PVDF membranes. The Snap i.d. system (Merck) was used for membrane blocking and antibody incubation. CD147 was detected with anti-CD147 antibody , phosphorylated eNOS was quantified with anti-phospho-eNOS (S1177) (1:1000) and its total levels were detected with anti-eNOS antibody (1:1000). NFkB-p65 was analysed with anti-NFκB-p65 (1:10000). CypA was detected with anti-CypA primary antibody (1:1000), CypB was recognized with anti-CypB antibody (1:1000), CypC was quantified with anti-CypC antibody (1:1000) and CypD was detected with anti-cyclophilin F primary antibody (1:1000). Protein band intensity was corrected using anti-β-actin (1:10000), anti-sodium/potassium ATPase (1:10000) and lamin B1 (1:1000) in cytosolic, membranous and nuclear fractions, respectively. The immunoreactive bands were detected with Supersignal West Pico Luminiscent Substrate and Supersignal West Femto Maximum Sensitivity Substrate. Diversity GeneSnap system and software  were used for protein bands detection. Experiments were performed three independent times by duplicate (n = 3). The specificity of anti-CypA, anti-CypB, anti-CypC and anti-CypD primary antibodies was checked before starting the experiments by loading 0.5 µg of human recombinant Cyps A, B, C and D in sodium dodecyl sulphate polyacrylamide gels and performing the protocol as described above.Electrophoresis was resolved in 4–20% sodium dodecyl sulphate polyacrylamide gels, containing 15 µg of cytosolic protein or 10 µg of membranous and nuclear fraction from each sample. Trans-BlotCypA, CypB, CypC and CypD  was used as normalization control , following the manufacturer’s instructions. RNA purity and concentration were determined with Nanodrop™ 2000 spectrophotometer (Thermo Fisher Scientific). cDNA was synthetized with 0.5 µg of RNA, oligo-dT primers and RevertAid Reverse Transcriptase, following manufacturer’s instructions. Quantitative PCR was performed using PowerUp SYBR Green Master Mix in a Step-One real-time PCR system . cDNA was amplified with specific primers for and CypD . Data we control . Relativp < 0.05, **p < 0.01 and ***p < 0.001.Results are presented as mean ± SEM. Differences were evaluated by one-way ANOVA and Tukey’s post hoc test with Graph Pad Prism 8.0 software. Statistical significance was considered at *At first, the effects of eCyps on cell viability were determined. MS1 cells were treated with eCyps A, B and C at 0.25 and 0.5 μg/ml, LPS at 1 μg/ml and CsA at 0.2 μM for 24 h and cell viability was evaluated by MTT test. None of the treatments affected cell viability , so the p < 0.001). Addition of eCypA at the highest concentration also induced a significant increase in IL-6 release of 15.3 ± 1.3 pg/ml (p < 0.05). Treatment with eCypB did not exert any effect on the cytokine, but eCypC at 0.5 μg/ml produced an increment in IL-6 levels . When endothelial cells were pre-treated with 0.2 μM CsA for 1 h before stimulation with eCypA and eCypC, the augmentation in IL-6 levels was significantly reduced up to 8.3 ± 1.3 and 11.8 ± 3.9 pg/ml, respectively (p < 0.05) (p < 0.01). Once again, eCypB did not produce an effect on the levels of the chemoattractant mediator. Pre-treatment with CsA significantly reversed the increase in MCP-1 generated by 0.5 μg/ml eCypA until 1684.8 ± 150.2 pg/ml, and reduced the levels of this chemokine after stimulation with eCypC at 0.25 and 0.5 μg/ml (829.7 ± 1542.4 and 1505.0 ± 501.5 pg/ml)  . MCP-1 w5 pg/ml) .p < 0.05) was observed when this immunophilin was added. This increase was reduced by pre-treatment with CsA  (p < 0.05). Pre-treatment with CsA decreased CD147 surface expression until 102.9 ± 3.8%   . When eCh eCypA) . Finallyh eCypC) .p < 0.05), which was counteracted when cells were treated with CsA . As expected, eCypB did not modify CD147 expression (p < 0.05). CsA reversed this effect, reducing CD147 levels up to 24.6 ± 5.4%   .p < 0.01). In this case, eCypB affected eNOS activation, reducing it until 45.3 ± 9.0% (p < 0.05). As expected, pre-treatment with CsA did not revert the inactivation produced by eCyps A and B due to the effects of the drug alone in the enzyme (p < 0.01)  .p < 0.05), whilst CsA diminished NFkB activation (p < 0.05). eCypA augmented the transcription factor translocation to the nucleus (119.3 ± 30.1%), which was reduced up to 79.5 ± 18.5% by pre-treatment with CsA, but both effects did not reach statistical significance. On the other hand, eCypB decreased NFkB activation, showing levels of 58.9 ± 10.4% and 78.9 ± 4.3%, without and with CsA, respectively (p < 0.05) in NFkB activation was observed, which was reverted by pre-treatment with CsA (p < 0.05)  .p < 0.05) (p < 0.05) (p < 0.05)  detected both CypD and CypA. Nevertheless, due to the different molecular weight of CypA and CypD , it was possible to distinguish both protein bands without uncertainty . Therefo < 0.05) . Surpris < 0.05) . In the  < 0.05) . When in < 0.05) , a reduc < 0.05)  and eCyp < 0.05) . Pre-trep < 0.01) and 23.7 ± 16.7% (p < 0.001), respectively (p < 0.01). Pre-treatment with 0.2 μM CsA produced a decrease to 154.5 ± 18.5%, but it was not statistically significant. Finally, intracellular CypD (iCypD) expression was monitored in presence of eCyps A, B and C (Regarding intracellular CypC (iCypC) expression, the positive control CsA reduced the levels of this immunophilin in MS1 cells . eCypA aectively . CsA sli B and C . LPS and B and C . With re B and C .CypA expression, agreeing with western blot results. Pre-treatment with CsA reduced the effect of both eCyps, although statistical significance was only reached with the combination of eCypB and the immunosuppressant drug (p < 0.05). With respect to CypB, a significant increase was found in this gene expression when LPS and eCypC were added (p < 0.05), and CsA counteracted the effect of the immunophilin (p < 0.01)    . CypC ex < 0.01) . Finally < 0.05) .Cardiovascular diseases and diabetes mellitus are non-communicable illnesses that cause 19 million of deaths each year, being cardiovascular pathologies the first cause of mortality worldwide . DiabetiThis work describes for first time the ability of eCypC to induce endothelial dysfunction. The protein produced an increase in the secretion of pro-inflammatory mediators and in CD147 expression, reducing eNOS activity and activating NFkB translocation to the nucleus . eCypC aBased on the obtained results, it can be presumed that eCypC activity is mediated by the receptor CD147, since the analysis of its expression revealed an augmentation in the plasmatic membrane. Cyps present a highly conserved active pocket and direct interactions among Cyps A and B and CD147 have been demonstrated, so CypC is probably acting similarly. In fact, CypC and CD147 have been co-localized in breast epithelial cells . AlthougRegarding eCypA, its effects on endothelial cells have been previously reported , but to eCypB showed different effects on pancreatic microendothelial cells than those produced by eCyps A and C. The role of CypB in inflammation is controversial as opposite results have been reported. eCypB is a well-known chemotactic factor for leukocytes , but itsCypA was downregulated after treatment with eCyps A and B, suggesting that both Cyps could have common signalling pathways. It has been described that reactive oxygen species (ROS) increase eCypA release in a ROCK2-dependent manner and high levels of eCypA stimulate ROS production, thus generating a vicious circle of increased oxidative stress  opening. The pore is a regulator channel of mitochondrial homeostasis that under pathological conditions is massively opened and leads to cell death . mPTP moThe crosstalk observed among eCyps addition and the alteration of their own intracellular expression, as well as other isoform levels, points to common pathways involved in the modulation of this protein family that should be further studied to better clarify the complex role of these chaperones in cell physiology.In conclusion, this work is the first study that allows a direct comparison between the three eCyps in the same cell line, as well as the first description of eCyps B and C effects on endothelial cells. Results indicate that eCypC acts similarly than eCypA, inducing pro-inflammatory mediators release, activating CD147 and generating endothelial dysfunction. eCypB presented different outcomes, without affecting neither the receptor nor cytokines release. Finally, it is evidenced a relationship among eCyps and iCyps levels, providing new insights into the intricate function of these proteins. These results in pancreatic microendothelial cells strengthen the crucial role of Cyps in the vascular dysfunction observed in diabetes, pointing to CypC as a potential mediator in this pathology.


The dopant of CePO4 could influence the activity of Au/GdPO4 due to the synergistic effect. GdPO4 and CePO4 nanorods were obtained by a hydrothermal process and the Au/GdPO4-rod and Au/Ce-GdPO4-rod catalysts were prepared by deposition–precipitation synthesis. The samples were extensively characterized by transmission electron microscopy (TEM), inductively coupled plasma (ICP), powder X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), ultraviolet-visible spectroscopy (UV-Vis), Fourier transform infrared spectroscopy (FT-IR), temperature programmed desorption  and N2 adsorption–desorption. The results showed that Au/GdPO4 with a low Au content possessed good activity for CO oxidation. When the content of Ce is 25 at%, 0.5% Au/Ce-GdPO4-rods can convert CO completely at 65 °C, and the catalyst showed better high-temperature resistance than 0.5% Au/GdPO4-rods. 0.5% Au/Ce-GdPO4-rods also showed good stability at reaction temperatures of 55 and 65 °C with CO conversions of 90% and 100% after continuous operation for 12 h. They also showed no deactivation after 50 h at a relative high reaction temperature of 200 °C.In this work, Au/GdPO 4–GdPO4 composite was prepared by a general ultrasound method and could be a good support for gold nanocatalysts. Au/CePO4–GdPO4 catalysts with a low content of gold showed good catalytic activity, high temperature resistance and stability for CO oxidation.A CePO Catalytic CO oxidation is still one of the most effective methods for the elimination of CO.6 Common CO oxidation catalysts mainly involve metal oxide, noble metal, and alloy catalysts.1,5,10 Supported gold nanoparticles have been well studied for CO oxidation as one of the most active catalysts due to their excellent catalytic performance.14–19CO oxidation is one of the most extensively investigated reactions in heterogeneous catalysis due to its importance in both practical applications and fundamental studies, such as in CO removal from exhaust gas, environmental protection and air cleaning, and as probe reaction for the demonstration of catalytic mechanisms.18–26 Many materials such as metal oxides and metal salts have been used as supports for noble metal catalysts, typically for Au catalysts.14,19,27 Most of them could not stand high temperature and have low stability. As known, the catalysts sometimes should be used at high temperature, so the catalyst must have high temperature resistance and stability. The deactivation and low stability have been the biggest drawbacks for Au catalysts' practical applications.28,29 How to develop feasible catalysts with excellent catalytic activity and stability for CO oxidation is still waiting to be solved. The major reason of the deactivation is often attributed to the agglomeration of Au nanoparticles, formation of carbonates adsorbed on the active sites, and change in oxidation state of Au species.26,29 References suggested that oxides or binary mixed oxides such as rare earth-TiO2,30 could help stabilize Au nanoparticles, which could prevent Au nanoparticles from sintering after deposition on the supports.26–33 More importantly in the composites, the advantages of the different phases could be combined to obtain strong surface interactions.3,7,11,14,34,35 It has been well established that the composition and structure of catalyst support play an important role in the catalytic performance. Tang et al. also found that hydroxyapatite/titanium-dioxide could stabilize gold nanoparticles due to the strong metal-support interaction.33 It significantly lowers the barrier to practical applications of supported Au catalysts, especially for high-temperature catalytic reaction.For a long time, Au has been considered chemically inert and inactive in catalysis. However, since Haruta demonstrated that when Au was supported on a certain metal oxides, it showed high CO oxidation catalytic activity, Au nanocatalysts have triggered a great deal of research activities in the past decades.4 nanorods, LnPO4  and nanosized YPO4 with low content of Au were extremely active for CO oxidation.36–38 However, the Au/YPO4-rods suffered severe deactivation after high temperature pretreatment. Therefore, we designed to use mixed CePO4–GdPO4 composite as supports via the synergistic effect between CePO4 and GdPO4 to obtain a type of high stable and sintering resistant Au supported catalyst.Recently our group also found that gold nanoparticles supported on BiPO4 and GdPO4 nanorods were prepared by hydrothermal process. CePO4–GdPO4 composite was prepared by a general ultrasound method. By a deposition–precipitation process, gold catalysts were successfully prepared. The techniques of inductively coupled plasma (ICP), N2 adsorption–desorption, powder X-ray diffraction (XRD), transmission electron microscope (TEM), X-ray photoelectron spectroscopy (XPS), temperature programmed desorption  and ultraviolet-visible spectroscopy (UV-Vis), Fourier transform infrared spectroscopy (FT-IR) were used for catalyst characterization. CO oxidation was selected as probe reaction to discuss the catalytic activity. The addition of CePO4 has a positive effect on the stabilization of gold particles, which helped the catalysts exhibit high catalytic activity, sintering resistance, and high stability.In this work, CePOAll chemicals in this paper were of analytical grade, and they were used directly without any further purification.4 nanorods were synthesized by a hydrothermal method used in 4H2PO4 (30 ml) and Ce(NO3)3 (30 ml) with molar concentration of 0.3 mol L−1 were mixed by vigorous stirring. 25 wt% ammonia was used to adjust the pH of the mixture to ∼2. Then the suspension was poured into a Teflon-lined stainless steel autoclave and kept at 160 °C for 14 h. After air-cooled to room temperature, the products were filtered, washed with deionized water and absolute alcohol. Finally, the white precipitate was dried at 80 °C overnight, then calcined at 400 °C for 3 h in air to get CePO4 nanorods.The CePO4 nanorods were synthesized by co-precipitation process. The calculated amounts of Gd(NO3)3·6H2O were mixed with 100 ml water–ethylene glycol solution (V2OH : VEG = 40 : 60). The obtained mixture was vigorously stirring for 1.5 h at 80 °C. Then 10 ml solution of NH4H2PO4 (0.05 g ml−1) was added to the above mixture. After that the suspension was refluxed at 140 °C for 3 h. Then the precipitate was washed with water and ethanol several times by centrifugation, dried at 80 °C overnight. The final white precipitate was calcined at 500 °C for 4 h in air with a heating rate of 5 °C min−1 to obtain GdPO4 nanorods.The GdPO4–GdPO4 composites  were obtained by an ultrasonic process. CePO4 and GdPO4 were mixed by milling 30 minutes. Then the mixture was dispersed in 100 ml H2O. After stirring 1 h, the suspension was then treated by ultrasonic dispersion for 2 h. Finally, the precipitates were centrifugated and dried at 80 °C overnight. The products were denoted as Ce0.05-GdPO4, Ce0.25-GdPO4, Ce0.50-GdPO4 with the concentration (in at%) of Ce as 5 at%, 25 at% and 50 at% respectively.CePO4 (0.01 mol L−1) was added with the nominal content of Au as 0.1%, 0.3% or 0.5%. Then via an ultrasonic process, the supports were distributed well in the solution of HAuCl4. Afterwards, certain amount of CO(NH2)2 was added to adjust pH value of the above suspension to ∼9. Then the suspension was heated in water bath at 90 °C for 4 h. At last, the precipitate was centrifuged, and washed with deionized water several times. Finally, the obtained Au catalysts were calcined at 300 or 500 °C for 2 h in air with a heating rate of 5 °C min−1. The concentrations of Au were expressed as percentage by weight percent.In the preparation procedure of all Au catalysts, Au was loaded by deposition–precipitation method. Firstly, 0.4 g supports were dispersed in 150 ml deionized water with stirring. After 2 h, amount of HAuClλ = 0.154 nm) in the 2θ range of 3–80° to check the crystallographic phase purity of the samples. Transmission electron microscope (TEM) observations were obtained with a JEM-2100 or JEM-2010FEF transmission electron microscopes operating at 200 kV. X-ray photoelectron spectroscopy (XPS) data were recorded on a Kratos Axis Ultra DLD X-ray photoelectron spectrometer using a monochromated Al Kα source operated at 150 W to identify the chemical composition and the oxidation state of the catalysts. Gold loadings of the catalysts were determined by inductively coupled plasma-atomtic emission spectroscopy (IRIS Intrepid II XSP). Ultraviolet-visible spectroscopy (UV-Vis) spectra were recorded on a UV-3600 UV-Vis NIR spectrophotometer. Fourier transform infrared spectroscopy (FT-IR) spectra of the samples were obtained with a FTS 6000 spectrophotometer. Temperature-programmed desorption (TPD) data were recorded in a Quantachrome ChenBet TPR/TPD. 100 mg of sample was heated to 300 °C in He for 1 h. After cooling to room temperature, the samples were exposed to corresponding gas  for adsorption until saturated. The relative desorption  was performed in 15 ml min−1 He with a heating rate of 10 °C min−1 from 50 to 800 °C. A cold trap prior to the thermal conductivity detector (TCD) was used during the desorption processes. The desorbed CO, O2 or CO2 was measured by thermal conductivity detector.The powder X-ray diffraction (XRD) study was carried out on a Rigaku D/Max-2500 X-ray diffractometer (kα 2 adsorption at liquid N2 temperature (77 K) using a Micromeritics apparatus. The samples were degassed at 473 K for 3 h prior to the adsorption experiments. The pore size was obtained from the adsorption branch of N2 isotherm by the BJH model.The Brunauer–Emmett–Teller (BET) surface areas of samples were measured by N−1. After holding at the reaction temperature for 30 min, the gaseous products were onlined analyzed by COx analyzer (GC-508A gas chromatography). This evaluation method of catalytic activity was similar to that described by our group elsewhere.36–38Catalytic activity evaluation was performed in a fixed-bed flow millireactor with an inner diameter of 8 mm. 200 mg of catalyst were diluted with 17.6 g chemically inert quartz sand. Subsequently, a mixture, 10% CO balanced with air was introduced into the reactor at a total flow rate of 36.3 mL min4-rods, Au/Ce-GdPO4-rods, and Au/CePO4-rods catalysts. The results suggested that Au loading efficiency of all catalysts was >70%. It almost reached 98% for 0.5% Au/CePO4-rods. After the addition of CePO4, Au loading efficiency increased. It revealed that Au species were more likely deposited on the surface of Ce-GdPO4 and CePO4. The synergistic effect could exist in Au/Ce-GdPO4. It is known that the Au-support interface largely depending on the deposition of Au particles is supposed to be an important factor for catalytic activity.4 Thus, the deposition of Au can suggest the interaction between support and Au species. Here, from the results, it could be concluded that CePO4 could help enhance electrostatic attraction leading to high gold deposition. The results revealed that the metal support interaction was strengthened by doping CePO4.4 and Au/GdPO4  calcined at 300 °C, the main diffraction peaks observed at 14.8°, 20.4°, 25.8°, 29.8°, 32.0°, 38.6°, 39.8°, 42.4°, 42.8°, 47.8°, 49.5°, 53.0°, 54.3°, and 55.3°could be attributed to (100), (101), (110), (200), (102), (112), (210), (211), (003), (301), (212), (203), (302), and (310) planes of hexagonal GdPO4 (JCPDS 39-0232). If the Au content was too low, it is not very accurate to analyze the samples by XRD. After deposition of Au, for comparison, 4-rods. As predicted, there was no peaks belonged to metallic Au (2θ = 38.2 and 44.5°).17 This could be due to low content and/or small particle size of gold nanoparticles which were lower than the detection limit of XRD. Thus, the Au size estimation could be obtained from TEM data. The X-ray powder diffraction patterns of CePO4–GdPO4 and GdPO4 supports are shown in 4, the reflections at 2θ = 25.2°, 29.2°, 38.9°, 46.7°, and 52.0°, were clearly assigned to hexagonal phases of CePO4. After the catalysts calcined at 500 °C , and the supported gold catalysts calcined at 300 °C and 500 °C for 2 h were characterized by N2 adsorption–desorption technique. As shown in 4 It could be seen in 4 nanorods and Ce-GdPO4 nanorods changed, suggesting the block dispersed Au nanoparticles into the pores of the supports. With increasing the calcination temperature, the surface area of the catalysts decreased which may be due to the agglomeration of Au nanoparticles after high calcination temperature pretreatment. While Au/Ce-GdPO4-rods exhibited larger surface area than Au/GdPO4-rods, indicating that Au/Ce-GdPO4-rods was more sintering-resistant with smaller Au nanoparticles than Au/GdPO4-rods after calcination at 500 °C. This result could be also proved by the TEM data. Yet, the high surface area and pore volume were beneficial for providing more active sites enhancing catalytic activity the catalysts.22,28,31 As shown in Fig. S1,4 nanorods possessed the BET surface area of 27 m2 g−1. It is noteworthy that Ce-GdPO4 nanorods obtained higher surface area than pure GdPO4 and GdPO4. The above results showed that synergistic effect might exist in Ce-GdPO4 nanorods. It could be also concluded that due to the synergistic effect of CePO4 between gold nanoparticles and the support in Au/Ce-GdPO4-rods catalysts, Au/Ce-GdPO4-rods calcined at elevated high temperature showed larger surface area than Au/GdPO4-rods, which would help enhance the activity. Combined with ICP data, we could conclude that this strong interaction in Au/Ce-GdPO4 would increase adsorption capacity of Ce-GdPO4 for Au species. Thus, Au loading efficiency was increased and higher than Au/GdPO4 enhancing the activity.The surface area and porosity of GdPO4 presents the morphology of rods with the width of 0.5–1.0 μm and the diameter of 8–15 nm. After the addition of CePO4, a homogeneous composite of CePO4 and GdPO4 nanorods was obtained -TEM (HRTEM) images were carried out. In obtained . The latobtained  of CePO4inset of  assigned 3.84 nm  highly d 4.03 nm , which w25-GdPO4 . However 5.91 nm , which wexpected . After to 8.0 nm . Howevert 5.0 nm . The exp2-TPD profiles of GdPO4 nanorods and Ce-GdPO4 composites, and the supported gold catalysts calcined at 300 °C. It can be seen that all the samples showed TPD peaks at 200–500 °C. After Au was deposited, a new desorption peak appeared at about 600 °C, indicating new active sites created in the Au supported catalysts. For pure GdPO4, a sharp TPD peak at ∼350 °C was detected. It could be found that after CePO4 was added, the desorption temperature of O2 shifted to high temperature for Ce0.05-GdPO4 nanorods. With increasing Ce contents, another weak desorption peak gradually appeared at about 600 °C attributable to representative of the desorption of strongly absorbed surface oxygen. It could be deduced that the modification of CePO4 to GdPO4 provided new O2 adsorption sites for the adsorption and activation of oxygen. The larger desorption peak, the stronger adsorption capacity of the sample to O2 and the more active sites. In CO catalytic oxidation process, CO molecules are usually adsorbed on the surface of Au. O2 is adsorbed on the surface of support or Au-support interface. The O2 desorption area increased in the order of Ce0.50-GdPO4 > Ce0.25-GdPO4 ≈ Ce0.05-GdPO4 > GdPO4, which indicated that the amount of surface-active oxygen species in Ce-GdPO4 is larger than that of the same species in GdPO4.6,27 This might be related with the interaction between CePO4 and GdPO4. It might suggest that the addition of CePO4 has a significantly influence on the formation of O2− species on the surface of support. 4 could supply active oxygen vacancies which may promote CO oxidation. This was consistent with TPD results of Au/Ce-GdPO4-rods in this work, which means that the addition of CePO4 could promote the generation of active oxygen. CePO4 could promote the generation of active oxygen, confirming the synergistic interaction in the Ce-GdPO4 composite. After the deposition of Au, the position of the TPD peaks for 0.5% Au/Ce0.25-GdPO4-rods and 0.5% Au/GdPO4-rods calcined at 300 °C shifted to higher temperature than their support. It revealed that the deposition of Au gives rise to O2-adsorption sites at metal-support interface for the adsorption and activation of O2 molecules. This result could be also confirmed with the TPD data of the catalysts calcined at 500 °C. Due to the synergistic effect in Au/CePO4-GdPO4-rods, fluidity of active O was enhanced, leading to the shift of desorption temperature, which is beneficial to CO oxidation. The results showed that CePO4 can interact with GdPO4 promoting the formation of active sites. The deposition of Au made the desorption of O2 more difficult. It means that the adsorption capacity of the catalyst for O2 is increased, and the activity of the catalyst may be changed.2-TPD Profiles of samples in 2 desorption temperature followed the order of Ce0.25-GdPO4 > 0.5% Au/GdPO4-rods > GdPO4 > 0.5% Au/Ce0.25-GdPO4-rods. Comparing with 0.5% Au/Ce0.25-GdPO4-rods, 0.5% Au/GdPO4-rods catalysts showed higher desorption temperature at ∼420 °C. This was an important indication of CO2 desorption ability of the samples, showing the major modifications of CePO4 and Au for their interaction. The strong adsorption of CO2 usually lowers the activity in catalysis.27 As shown in Fig. S4,2 absorbed on Au/CePO4 relatively, which might facilitate the good stability of the catalysts. The CO2 adsorption capacity of these catalysts was different from each other indeed. It would be synergistic interaction of CePO4 led to fewer basic sites and lower basicity of 0.5% Au/Ce0.25-GdPO4-rods compared to 0.5% Au/GdPO4-rods. From the results we can conclude that there were more basic sites in 0.5% Au/GdPO4-rods than in 0.5% Au/Ce0.25-GdPO4-rods. CO2 is more easily absorbed on 0.5%Au/GdPO4-rods than 0.5% Au/Ce0.25-GdPO4-rods. Thus, CO2 would desorb from active sites more difficultly in 0.5% Au/GdPO4-rods than in 0.5%Au/Ce-GdPO4-rods leading to the easy production of carbonate species, which might result in the deactivation of the catalysts.27Similar conclusion was obtained with CO4-rods and Au/Ce-GdPO4-rods are shown in 4-rods are sharper than Au/GdPO4-rods indicating the stronger CO adsorption ability than Au/GdPO4-rods. Furthermore, in the range of 550–650 °C, the desorption temperature and the peak area of Au/Ce-GdPO4-rods were much higher and larger than that of Au/GdPO4, also indicative of the stronger CO adsorption on the Au/Ce-GdPO4-rods catalyst. In general, the stronger the adsorption of CO, the more number of active sites the catalysts owned.7,13 It's clear Au/Ce-GdPO4-rods had more active sites than Au/GdPO4-rods. The above characterization results showed that synergistic effect might exist in Ce-GdPO4-rods.CO-TPD analysis was also chosen to investigate the ability of CO adsorption on the catalysts. The CO-TPD spectra of Au/GdPO3/2 spin–orbit states and 3d5/2 spin–orbit states. As defined in the picture, v, v′ centered at 882.1 and 885.8 eV represent the Ce 3d5/2 contributions, and u, u′ centered at 900.5 and 903.9 eV represent 3d3/2 contributions, which are the characteristic peaks for trivalent states of Ce3+. There was no distinct peaks detected at about 916 eV , which arises from a transition of the 4 f′′ final state from the 4 f′′ initial state, suggesting no existence of Ce4+.3,7,15,17,29 Since it is absent in Au/Ce-GdPO4, combining with The signals v′ and u′, it could be deduced that Ce is mainly present as Ce3+ in the samples. In 4-rods, Gd 4d peaks centered at 143.1 (Gd 4d5/2) and 148.2 eV (Gd 4d3/2) indicating that Gd ions are Gd3+.3,41,42 However, the values of binding energy of Gd 3d3/2 for 0.5% Au/Ce0.25-GdPO4-rods calcined at 300 and 500 °C were about 0.4 eV lower than that for Au/GdPO4, which could be assigned to Gd ions embedded in CePO4 nanorods due to the interaction of CePO4 with GdPO4. As shown in v) species which was the evidence for the formation of P–O chemical bonding in this report.33,39,40 The relatively lower binding energy of the P 2p for Au/Ce-GdPO4 calcined at 300 °C and 500 °C than that for Au/GdPO4 may also suggest that the Gd ions interact with CePO4. It is obviously concluded that CePO4 and GdPO4 were incorporated together due to the interaction of CePO4 with GdPO4.33 For the fresh 0.5% Au/GdPO4-rods and 0.5% Au/Ce0.25-GdPO4-rods, a broad O 1s XPS peak , Gd (+3), P (+5) and O (−2) species were simultaneously present in the samples. As shown in XPS peak  was foun4 and GdPO4. For the 0.5% Au/Ce0.25-GdPO4-rods catalysts, as shown in −1 were ascribe to asymmetric stretching and bend vibrations of PO43−.34,38,43 For the spent catalysts, there was no new absorption peaks appeared compared with the fresh 0.5% Au/Ce0.25-GdPO4-rods. However, in −1 for spent 0.5% Au/GdPO4-rods were indexed as the υ(CO3) of bicarbonate or bidentate carbonate species and absorbed CO compared with fresh 0.5% Au/GdPO4-rods.1,5,27 It revealed that there were carbonate species formed on the surface of Au/GdPO4 after combustion for CO oxidation. It has been reported that the accumulation of carbonate species could block the active sites leading to the deactivation of catalyst resulting in the deactivation of 0.5% Au/GdPO4-rods. As expected, 0.5% Au/GdPO4-rods showed lower stability than 0.5% Au/Ce0.25-GdPO4-rods in the reaction of CO oxidation.FT-IR spectra of the fresh and spent catalysts were presented in 4-rods and Au/Ce-GdPO4-rods were also confirmed by the UV-Vis spectra  were 90, 65, 50 °C, respectively. It was noteworthy that the Au/GdPO4-rods catalysts showed good activity for CO oxidation with low content of gold. After adding CePO4 to GdPO4  much smaller than 0.5% Au/Ce0.25-GdPO4-rods (1.77 s−1). It could be seen form the results of TEM that the diameter of Au particles in 0.5% Au/GdPO4-rods pretreated at 500 °C was about 10 nm which was much bigger than 7 nm for 0.5% Au/Ce0.25-GdPO4-rods. As known, the particle diameter of Au particles was a vital factor in determining the activity of gold catalysts. The BET results also indicated that after high temperature treatment, the surface area of 0.5% Au/GdPO4-rods decreased more than that of 0.5% Au/Ce0.25-GdPO4-rods. The TPD results also showed that 0.5% Au/Ce0.25-GdPO4-rods calcined at 500 °C could supply more active sites than 0.5% Au/GdPO4-rods. It was clear that these characteristics on Au/Ce-GdPO4-rods were different from those on Au/GdPO4-rods, which showed weak high-temperature resistance. These results might explain that 0.5% Au/Ce0.25-GdPO4-rods calcined at 500 °C possessed better activity than 0.5% Au/GdPO4-rods. The relative high catalytic performance 0.5% Au/Ce0.25-GdPO4-rods calcined at 500 °C were also comparing the activities calculated at 80 °C in the literatures which was similar to Au/TiO2 (2.8 s−1) and Au/Ce–K-OMS-2 (2.3 s−1).14,15 Indeed, it was the interaction between CePO4 and GdPO4 which fabricate the supported Au nanoparticles improving sintering resistance. And the Au active species on the interface of CePO4–GdPO4 made important contribution to the high-temperature resistance of Au/Ce-GdPO4-rods. So it could be concluded that the synergistic interaction of CePO4 could help stabilize Au nanoparticles and prevent the agglomeration of Au nanoparticles.2,22,26,27 The stability tests were also performed for 0.5% Au/GdPO4-rods and 0.5% Au/Ce0.25-GdPO4-rods in comparison to study the synergistic effect, maintaining a continuous CO oxidation reaction at temperature of 100% CO conversion for 12 h respectively. 4-rods showed apparent deactivation in 12 h. The CO conversion decreased with 0.5% Au/GdPO4-rods from 100% to 90% in the first 3 h, and after another 3 h, CO conversion was kept on decreasing to 40% deactivation. But the deactivation for 0.5% Au/Ce0.25-GdPO4-rods was different. It could be obviously found that in the 12 h of reaction time, CO conversion did not decrease. At the reaction temperature of 55 °C (0.25-GdPO4-rods decreased only 2% (from 90% to 88%) of its initial activity. After the calcinations at 500 °C .As known, not only the activity of supported gold catalysts is of great importance but also the stability.of 55 °C , after ct 500 °C , the 0.54, owning high loading efficiency and small particle size; (2) Au deposited on CePO4–GdPO4 interface, which possessing good activity and stability due to the strong synergistic effect between Au and Ce-GdPO4 or CePO4 in the catalysts; (3) Au on GdPO4, suffering from deactivation owning to the block of the active sites by carbonates resulting from the reaction. The first two functions made relative more contribution to catalytic CO oxidation than the third one depending on the promotional effect of CePO4.As discussed above, we could finally draw the conclusion that there may be three sorts of active component (1) Au supported on CePO4 composite was developed. The strong interaction of CePO4 in the composite could account for the sintering resistance and stability of the Au nanoparticles. The CO catalytic tests showed that the Au/GdPO4-rods catalyst with low Au content still has good catalytic performance. It's obvious that after high temperature calcination, Au/Ce-GdPO4-rods showed better activity than Au/GdPO4-rods. And Au/Ce-GdPO4-rods also possessed good durability. CePO4 could effectively improve sintering resistance and stability of Au nanoparticles in the catalyst because CePO4 as auxiliary for GdPO4 could make Au fixed on the surface of the support, and inhibited the aggregation of Au nanoparticles. In all, Au/GdPO4-rods could be good catalysts with low content of Au, but the catalyst showed bad sintering resistance to high temperature and poor durability. However, the designed Au/Ce-GdPO4-rods catalyst by a grinding-ultrasonic method possessed good catalytic activity, good sintering-resistance and excellent stability. The enhancement of activity by the dopant of CePO4 is related to synergistic effect between CePO4 and GdPO4 or Au and the support. This physical mixing method is an effective procedure. In addition, it significantly has the value of practical applications.In summary, a new Ce-GdPOThere are no conflicts to declare.RA-008-C8RA02206B-s001


Moringa oleifera leaf meal (MOLM)  and fat  on fatty acid profile, lipid oxidation, and proximate composition of chicken droëwors. On triplicate samples of all treatments, proximate analysis was done, the total lipid was quantitatively extracted using chloroform and methanol in a ratio of 2 : 1, fatty acid profiles were determined, and thiobarbituric acid reactive substances (TBARS) were measured. The present study showed that droëwors manufactured with 0% fat inclusion had less fat and more protein than those made with 10% and 15% fat. All treatments contained a greater percentage of C18:1c9 (oleic) (30.95 to 32.65%) acid than other fatty acids and a higher proportion of unsaturated fatty acids than saturated. T9  had significantly (P < 0.05) higher PUFAs than T1  and T4 . Treatments with 0.5% MOLM had significantly lower TBARS values after drying (0.01-0.07 mg MDA/kg) than treatments with 0% and 2.5% MOLM (0.05–0.15 mg MDA/kg). Therefore, MOLM inclusion at 0.25 and 0.5% effectively decreased TBARS of chicken droëwors with up to 15% fat inclusion after 72 h of drying and 168 h of storage and is a potentially good source of natural antioxidants for this traditional dried sausage product.We present the first report on the effect of graded levels of  Droëwors is a dried traditional South African salted sausage product typically made using beef or a variety of game meats . ChickenMoringa oleifera grows naturally in various countries. It has multiple names, such as the drumstick tree, ben oil, and horseradish [Moringa oleifera plant possesses numerous multifunctional applications in human nutrition and animal nutrition and in the processing of animal food products [Moringa oleifera leaves are high in nutrients such as high potassium, iron, protein, and vitamin C [Moringa oleifera is a powerful natural antioxidant since it contains flavonoids, tocopherols, vitamin C, and other phenolic compounds [seradish . This plseradish . Originaproducts . Moringaitamin C . This plitamin C . Moringaompounds .Moringa oleifera leaf meal (MOLM) to chicken droëwors with different fat levels to prevent lipid oxidation.Consumers are more careful about the food quality, especially the nutritional attributes. Fatty acid profiles are among the dietary characteristics that consumers are concerned about . In meatEthical clearance  was obtained from the University of Fort Hare Research Ethics Committee before the research commenced.Lean chicken meat, chicken fat, dehydrated natural sheep casings , salt, and ground black pepper were purchased for droëwors preparation. The lean meat and fat were trimmed into 5 × 5 cm cubes and were divided into nine (9) batches and nine (9) treatments. The treatments were prepared with different combinations of fat and MOLM . The chiAfter drying (72 h), triplicate samples (±50 g each) of each treatment were analysed for proximate composition. Moisture (Method 934.01) and ash (Method 924.05) contents of the dried droëwors were determined according to the methods defined in . The prow/w) per 100 g tissue.After drying (72 h), triplicate samples (±50 g each) of each treatment were analysed for fatty acid composition. Total lipid from dry sausage was quantitatively extracted, as described in the literature, using chloroform and methanol in a ratio of 2 : 1 . Antioxiw/w) per 100 g tissue by determining the difference in weight. The muscle moisture content and methanol BF-3 were determined by subtraction (100% − %lipid − %FFDM) and expressed as % moisture (w/w) per 100 g tissue. The extracted fat from dry sausage was stored in a poly-top  under a blanket of nitrogen and frozen at -20°C pending fatty acid analyses.The fat-free dry matter (FFDM) content was determined by weighing the residue on a preweighed filter paper used for Folch extraction after drying. The FFDM was expressed as % FFDM (μm film thicknesses). The analysis was performed using an initial isothermic period (40°C for 2 minutes). Thereafter, the temperature was increased at a rate of 4°C/minute to 230°C. Finally, an isothermic period of 230°C for 10 minutes followed. FAMEs n-hexane (1 μL) were injected into the column using a Varian CP 8400 Autosampler. The injection port and detector were both maintained at 250°C. At 45 psi, hydrogen functioned as the carrier gas, while nitrogen was employed as the makeup gas. Galaxy Chromatography Software recorded the chromatograms.A lipid aliquot (20 mg) of dry sausage lipid was transferred into a Teflon-lined screw-top test tube using a disposable glass Pasteur pipette. Fatty acids were trans-esterified to form methyl esters using 0.5 N NaOH in methanol and 14% boron trifluoride in methanol . FAMEs fFatty acid methyl ester samples were identified by comparing FAME peaks' retention times from samples with those of standards obtained from Supelco . All other reagents and solvents were of analytical grade and obtained from Merck Chemicals . Fatty acids were expressed as the proportion of each individual fatty acid to the total of all fatty acids present in the sample. The following fatty acid combinations were calculated: omega-3 (n-3) fatty acids, omega-6 (n-6) fatty acids, total saturated fatty acids (SFA), total monounsaturated fatty acids (MUFA), polyunsaturated fatty acids (PUFA), PUFA/SFA ratio (P/S), and n-6/n-3 ratio.3)) and 12.5 mL distilled water for 180 sec using a Stomacher 400 Laboratory Blender . Slurries were filtered (0.45 μm), and duplicate samples of filtrate (3 mL) were added to an equal volume of 0.02 M 2-thiobarbituric acid. An equal volume of distilled water was added to the third replicate to act as a turbidity blank for each sample. Samples were vortexed for 10 sec, incubated in a water bath at 70°C for 1 hour until pink colour development, and allowed to cool for 30 min, and the absorbance was recorded at 532 nm. TBARS were calculated using 1,1,3,3-tetraethoxypropane (TEP) as a standard. Results were expressed as mg of malonaldehyde (MDA) equivalents/kg of meat.For analysis of the evolution of lipid oxidation, sampling of chicken droëwors was implemented in triplicate (±50 g each) from each batch at 0, 0.25, 0.5, and 72 h during drying. The droëwors were stored, unpackaged, and uncovered in a ventilated room under ambient conditions and sampled after 7 days (168 h). The content of thiobarbituric acid reactive substances (TBARS), determined at 0, 0.25, 0.5, 72, and 168 hrs, was performed using the technique described in the literature . BrieflyP < 0.05. The statistical model used is as follows:Yijkl is a dependent variable , μ is the overall mean, αi is the effect of fat , βj is the effect of Moringa oleifera leaf meal , αβγijk is the interaction (between fat and MOLM at different levels of inclusion), and eijkl is random error.The data on lipid oxidation, proximate composition, and fatty acid profile of chicken droëwors were analysed using the PROC GLM procedures of , and paiP < 0.05) ash content than T4 and T5. There were no significant differences in moisture content across treatments. T1-T3 had higher protein (63.25 ± 1.65 to 67.56 ± 2.33%) than T4-T6 (58.30 ± 1.65 to 59.40 ± 1.65%) and T7-T9 (45.80 ± 1.65 to 54.16 ± 1.65%). T6 had significantly higher ash content (14.92 ± 0.65%) than T4 (11.36 ± 0.65%) and T5 (12.45 ± 0.65%). However, this increasing trend was variable or insignificant across other treatments with the same fat inclusion levels and increasing MOLM levels.Results of protein, moisture, fat, and ash for each treatment are presented in P < 0.05) TBARS than droëwors with 0%  and 0.25% . After 168 h of storage, TBARS in T1 (0.12 ± 0.01 mg MDA/kg), T4 (0.13 ± 0.01 mg MDA/kg), and T7 (0.10 ± 0.00 mg MDA/kg) were significantly higher than T2 (0.05 ± 0.03 mg MDA/kg), T3 (0.01 ± 0.00 mg MDA/kg), T6 (0.04 ± 0.01 mg MDA/kg), T8 (0.03 ± 0.02 mg MDA/kg), and T9 (0.05 ± 0.01 mg MDA/kg).P < 0.05) ash content than the treatments with 0 and 0.25% MOLM. This may be attributed to additional minerals in the MOLM, as they are reportedly a rich source of vitamins and minerals [Raw chicken has an average moisture content of 75% . The preminerals . Higher minerals . HoweverChicken droëwors in the present study had high percentages of palmitic acid, stearic acid, linoleic acid, and oleic acid . This fiThe results of the present study support that the chicken droëwors produced in the present study are suitable for consumer health. The literature further indicates that an increase in PUFA content plays an essential role in human health. Furthermore, high PUFA content confers economic benefits due to the high antioxidant content in MOLM on inhibiting lipid oxidation inhibition in chicken droëwors, as reported by Prisacaru . The droP > 0.05) across all treatments. The inclusion of 0.25% and 0.5% of MOLM caused a reduction (P < 0.05) in lipid oxidation of T6 and T9 after drying and 168 h of storage. The TBARS may have reduced due to the inhibition of lipid oxidation by MOLM. Moringa oleifera contains polyphenols with antioxidant effects. Plant phenolic compounds contribute to their antioxidative capacity by free radicals' scavenging capability, as reported by Falowo et al. [Moringa oleifera in the chicken sausages resulted in lower TBARS [α-tocopherol, β-carotene, ferric reducing antioxidant power, physicochemical properties, and lipid oxidation of dry pork sausages at processing and storage [Moringa oleifera leaf powder during drying and up to 10 days of storage. Like chicken droëwors, pork droëwors are reportedly more susceptible to lipid oxidation due to their high PUFA content [Before drying, TBARS values were similar  to chicken droëwors decreased lipid oxidation after 72 h of drying and 0.25-0.5% MOLM reduced lipid oxidation during 168 h of storage. There were high levels of stearic, palmitic, linoleic, and oleic fatty acids in chicken droëwors. With the predisposition of unsaturated fatty acids to lipid oxidation, the incorporation of MOLM as a natural antioxidant indicates that chicken meat could successfully be used to provide an alternative droëwors option for consumers.


Pdk4) were increased via co-treatment of butyrate and TNFα, suggesting the synergistic inhibition of glycolysis. TNFα alone reduced the expression of monocarboxylate transporters slc16a1 and slc16a3, suggesting effects of TNFα on butyrate uptake into colonocytes. Of the 185 metabolites that were detected with LC-MS, the TNFα-induced increase in biopterin produced the only significant change, suggesting an alteration in mitochondrial biogenesis in colonocytes. Considering the reports of elevated colonic TNFα and reduced butyrate metabolism in many conditions, including in hypertension, the present work sheds light on cellular interactions between TNFα and butyrate in colonocytes that may be important in understanding conditions of the colon.Butyrate, a short-chain fatty acid, is utilized by the gut epithelium as energy and it improves the gut epithelial barrier. More recently, it has been associated with beneficial effects on immune and cardiovascular homeostasis. Conversely, tumor necrosis factor alpha (TNFα) is a pro-inflammatory and pro-hypertensive cytokine. While butyrate and TNFα are both linked with hypertension, studies have not yet addressed their interaction in the colon. Here, we investigated the capacity of butyrate to modulate a host of effects of TNFα in primary rodent colonic cells in vitro. We measured ATP levels, cell viability, mitochondrial membrane potential (MMP), reactive oxygen species (ROS), mitochondrial oxidative phosphorylation, and glycolytic activity in colonocytes following exposure to either butyrate or TNFα, or both. To address the potential mechanisms, transcripts related to oxidative stress, cell fate, and cell metabolism  were measured, and untargeted liquid chromatography–tandem mass spectrometry (LC-MS/MS) was employed to profile the metabolic responses of colonocytes following exposure to butyrate and TNFα. We found that both butyrate and TNFα lowered cellular ATP levels towards a quiescent cell energy phenotype, characterized by decreased oxygen consumption and extracellular acidification. Co-treatment with butyrate ameliorated TNFα-induced cytotoxicity and the reduction in cell viability. Butyrate also opposed the TNFα-mediated decrease in MMP and mitochondrial-to-intracellular calcium ratios, suggesting that butyrate may protect colonocytes against TNFα-induced cytotoxicity by decreasing mitochondrial calcium flux. The relative expression levels of pyruvate dehydrogenase kinase 4 ( Gut dysbiosis contributes to a variety of conditions including irritable bowel disease, hypertension (HTN), and colon cancer, among others ,2. The cIn contrast, elevated TNFα leads to colonic cell shedding, impairment in colonic barrier integrity , and colThe current study examined the effects of increasing concentrations of TNFα and butyrate, individually and in co-treatment, on an array of cellular functions in rat primary colonocytes. The results of the current study improve our mechanistic understanding of the effects of TNFα and butyrate in the colon on mitochondrial metabolic endpoints and other cellular functions and shed light on the previously undocumented synergistic effects of butyrate and TNFα on colonic biopterin.® , AlamarBlue™ , CellTox™ Green , Mitochondrial Membrane Potential Kit , and H2DCFDA dye . The reagents and kits used in the experiments include sodium butyrate , rat TNFα , oligomycin A , 4-trifluoromethoxyphenylhydrazone (FCCP) , antimycin A , rotenone , glucose , and 2-deoxyglucose , which were all purchased from Sigma Aldrich. The microplate assays included CellTiter-GloThe experiments were performed in physiologically normal (Sprague Dawley) rodent colonic epithelial cells rather than human cells, due to inherent ethical issues with collecting physiologically normal colonocytes from humans. Our research focus is also on spontaneously hypertensive rodents as a model, and we aim to study corresponding responses in vivo in rodent models. Doses were selected to cover a broad range of physiological butyrate concentrations and both physiological and pathological colonic TNFα concentrations. Specifically, TNFα has been reported in the range of 50 pg/mL in Sprague Dawley rats and 150–1000 pg/mL in various rodent models of hypertension ,25. ButyPrimary rat colonic epithelial cells  were maintained in Complete Epithelial Media , passaged using Hanks Balanced Salt Solution (HBSS)  and TrypLE Express , and counted used a hemocytometer. Cells were seeded onto a black 96-well plate  at 10,000 cells per well in 100 µL of medium. The following day, cells were exposed in triplicate (6 wells for medium control) to increasing doses of TNFα (50 pg/mL up to 10 ng/mL), butyrate (50 µM up to 25 mM), as well as co-treatments of 500 µM butyrate and a subset of TNFα doses (50 pg/mL–1 ng/mL) by adding 100 µL of a 2× solution to each well. The lysis control supplied with the CellTox™ Green kit served as the cytotoxic positive control and was added 20 min prior to the measurement of cytotoxicity as a ½ dilution of a 2× stock. On the day of the assay, 100 µL was then removed prior to the addition of 100 µL of CellTox™ Green reagent using a multichannel pipettor. The 96-well plate was mixed via orbital shaker for 30 s at 400 rpm. The plate was then incubated at room temperature for 15 min before fluorescence was measured at an excitation/emission (ex/em) of 485/520 nm on a BioTek Synergy™ 4 Hybrid Microplate Reader using the autogain feature. Following this, 22.2 µL of AlamarBlue™ reagent was added to each well  and the plate was incubated at 37 °C for 2 h before an absorbance of 570 nm was measured using the same microplate reader. Wells containing media but no cells were used to remove the average of the background signal. The AlamarBlue™ and CellTox™ Green assay values were normalized to the negative control (containing media only). The data represent one technical replicate .® 2.0 reagent with a multichannel pipettor. The 96-well plate was mixed via orbital shaker for 30 s at 400 rpm. After 10 min of incubation at room temperature, the luminescent signal was recorded on a BioTek Synergy™ 4 Hybrid Microplate Reader using the autogain feature. The data represent the combination of two technical replicates normalized to the medium control .Cells were seeded onto a white 96-well plate for each technical replicate  at 10,000 cells per well in 100 µL of medium. The following day, cells were exposed in quadruplicate to increasing doses of TNFα (50 pg/mL to 10 ng/mL) and butyrate (50 µM to 25 mM) by adding 100 µL of a 2× solution to each well. As concentrations for TNFα in the literature are typically described as weight per volume , while butyrate is recorded using molar concentration , we report both in this format. On the day of the assay, 100 µL was then removed prior to the addition of 100 µL of CellTiter-Glo2 at 37 °C for 1 h, before 50 µL of reagent B was added to each well. The 96-well plate was mixed via orbital shaker for 30 s at 400 rpm. Fluorescence was measured at an ex/em of 540/590 (red) and 490/525 (green) on a BioTek Synergy™ 4 Hybrid Microplate Reader using the autogain feature. The ratio of red/green fluorescence intensities was calculated in Excel . Wells containing media but no cells were used to remove the average of the background signal. MMP assay values were normalized to the negative control (containing media only) by dividing everything by the average values for this control. The data represent the combination of two technical replicates normalized to the medium control .Cells were seeded onto a black 96-well plate at 20,000 cells per well in 100 µL of medium. The following day, the plate was exposed in triplicate to increasing doses of TNFα (50 pg/mL up to 5 ng/mL), butyrate (50 µM up to 7.5 mM), as well as co-treatment of 500 µM butyrate and TNFα (50 pg/mL–5 ng/mL) by adding 100 µL of 2× chemical stocks. FCCP is a protonophore that allows hydrogen to pass freely through lipid bilayers and served as a positive control for the loss of mitochondrial membrane potential (MMP). After 24 h of incubation, 100 µL was removed. JC10 dye solution A was prepared by adding 50 µL of JC10 dye to 5 mL of reagent A and vortexing. Next, 50 µL of this mixture was added to each well using a multichannel pipette. The plate was then mixed via an orbital shaker at 400 rpm for 30 s; then, it was incubated in 5% CO2 incubator. After 30 min of incubation with the calcium indicators, the 96-well plate was put into the machine for a 1 h kinetic run with reads every 5 min spanning the 24 h time period. The reads consisted of excitation/emission values of 552/581 nm for Rhod-2 and 490/525 nm for Fluo-5F, with automatic gain, measured as a bottom read. The background was removed from the data by subtracting the no-cell-fluorescence values from all groups. The data represent one technical replicate normalized to the medium control .Colon epithelial cells were seeded at 20,000 cells/100 µL/well in a black 96-well plate and allowed to adhere overnight. Cells were then exposed (n = 6 wells/treatment) for a 24 h time period by adding 100 µL of 2× reagent in media to each well. The treatments included media only, positive control, no cell wells, 500 µM, 1000 µM, 2500 µM, and 5000 µM butyrate, or 250 pg/mL, 500 pg/mL, 750 pg/mL, and 1000 pg/mL TNFα plus and minus 500 µM butyrate. The positive control included 5 µM FCCP, as the mitochondrial calcium indicator Rhod-2 is dependent on the mitochondrial membrane potential. A total of 50 µL of medium containing 5 µM Rhod-2 and 20 µM Fluo-5F  was then added to each well using a multichannel pipette. The final concentrations were 1 µM for Rhod-2 and 4 µM for Fluo-5F. Following the exposure and addition of calcium indicators, the 96-well plate was mixed via orbital shaker at 400 rpm for 30 s and returned to the 37 °C CO2 at 37 °C for 45 min. Fluorescence was measured at an ex/em of 485/535 nm on a BioTek Synergy™ 4 Hybrid Microplate Reader using the autogain feature. The data represent the combination of two technical replicates normalized to the medium control .Cells were seeded onto a black 96-well plate  at 25,000 cells per well in 100 µL of medium. The following day, the plate was exposed in triplicate to increasing doses of TNFα (50 pg/mL to 5 ng/mL), butyrate (50 µM to 7.5 mM), as well as co-treatment of 500 µM butyrate and TNFα (50 pg/mL–5 ng/mL) by adding 100 µL of 2× chemical stocks. Next, 50 µM menadione was added 4 h prior to the 24 h time period and served as a positive control for ROS production. After 24 h of incubation, 100 µL was removed. A 40 µM H2DCFDA dye solution (2×) was prepared and vortexed. Next, 100 µL of this mixture was added to each well using a multichannel pipette. The plate was mixed at 400 rpm via orbital shaker; then, it was incubated in 5% CO2 incubator for 1 h while the flux plate  was loaded with 10× injection chemicals. The Glycolytic Stress Test and Cell Mitochondrial Stress Test consisted of a constant concentration a 10× injection scheme. The Glycolytic Stress Test consisted of the sequential injection of 56 µL of 250 mM glucose, followed by 62 µL of 20 µM oligomycin, and finally, 69 µL of 1M of 2-deoxyglucose. The Cell Mitochondrial Stress Test consisted of the sequential injection of 10× chemicals, specifically 56 µL of 7.5 µM oligomycin, followed by 62 µL of 30 µM FCCP, and finally, 69 µL of 10 µM of antimycin A and rotenone. Cells were seeded onto a cell microplate  at 25,000 cells per well in 100 µL of medium. To prevent edge effects following seeding, cells were left at room temperature for 30 min prior to incubation at 37 °C. Following cell attachment in 6–8 h, 150 µL was added. The following day, the plate was exposed by adding an additional 250 µL of 2× chemical stocks. Following 24 h exposure, the cells were washed three times by removing, and then, adding 350 µL of warm Krebs Heinseleit buffer +200 µM glucose lacking sodium bicarbonate (pH 7.1 to 7.2 before sterile filtering). Following washes, the cells were transferred to a 37 °C non-COFive separate flasks of cells were treated with either butyrate (500 µM), TNFα (500 pg/mL), or co-treatment together (500 µM butyrate and 500 pg/mL TNFα) for 24 h as per the methods outlined above. The 500 pg/mL TNFα is within the range of serum concentrations for different hypertensive models, including the SHR (150 to 300 pg/mL) and the Sabra rat model of salt-sensitive hypertension (150 to 1100 pg/mL) , whereas® Supermix . Biological replicates  were measured in duplicate. Two reference genes  and beta actin (β-actin)) were used to normalize target expression. The M-value and coefficient of variation for the two combined reference genes were M = 1.34 (CV = 0.46) for cDNA synthesis 1 and M = 0.66 (CV = 0.23) for cDNA synthesis 2. Due to the number of transcripts assessed, two cDNA synthesis reactions were conducted. In each case, genes were normalized to rps18 and β-actin. Normalized expression based upon the relative ΔΔCq method was obtained for each target gene using CFX Manager™ software (v3.1) (baseline subtracted) [Target genes were selected based upon their role in mitochondrial oxidative respiration, glycolysis, cell differentiation, oxidative stress, and apoptosis. These genes included Pdk1, Pdk2, Pdk4, Spr, Slc16a1, Slc16a3, Lgr5, Casp3, Tnfr2, Bax, Bcl2, Cs, Ppargc1a, Sod1, Sod2, and Cat. tracted) . Real-titracted) .2 incubator, before the cells were transferred to a 15 mL conical flask with 4× the volume of HBSS. The groups of four were then centrifuged for 5 min at 300× g. The cell pellet was then aseptically resuspended in 1 mL of ice-cold rinsing solvents , transferred to a pre-weighed and labeled 1.7 mL microtube , and centrifuged at 300× g for 5 min. The supernatant was then discarded and the cells were transferred to a −80 °C freezer until extraction.Colon epithelial cells were exposed in T75 flasks for 24 h to either medium, 500 µM butyrate, 500 pg/mL TNFα, or a combination of butyrate and TNFα (N = 8/group) by removing the media and replacing them with 8 mL of 1× chemical stocks. Following this exposure, cells were passaged in groups of 4  by removing the media and washing the flask with 10 mL of sterile Hank’s Balanced Salt Solution (HBSS), then with 3 mL of TrypLE Express for 2 min in a 37 °C COv/v/v; Optima grade) and vigorous vortexing for 30 min. The samples were centrifuged at 15,000× g at 4 °C for 30 min. The supernatants were carefully extracted, transferred into new tubes, and evaporated fully under a stream of nitrogen gas at 30 °C. Dried metabolite extracts were reconstituted in 100 µL 95:2:2:1 water:methanol:acetonitrile:formic acid (v/v/v/v) and were saved at 4 °C until analysis.Medium-free cell pellets (approximately 50 mg/sample) or quality control samples  were thawed on ice. Known amounts of isotopically labeled internal standards in water with 0.1% formic acid  were added to each sample, followed by the addition of 1200 µL ice-cold (−20 °C) 8:1:1 acetonitrile:methanol:acetone  were performed using pooled samples created by mixing equal parts of extracts from each sample for each experimental condition. IE-Omics [Metabolomic analyses were completed using a Thermo Vanquish UHPLC system coupled to a Thermo Q-Exactive Orbitrap mass spectrometer. A Waters Acquity HSS-T3 column was used (150 mm × 2.1 mm × 1.8 µm), and the mobile phase consisted of (A) 100% water and (B) 100% acetonitrile, both with 0.1% formic acid. The multi-step gradient was as follows: Starting at 1% B, hold until the 3 min mark; then, linearly increase to 50% B between 3 and 11 min, followed by a linear increase to 98% B between 11 and 12 min. Hold at 98% B between 12 and 16 min, decrease to 1% B at 16.1 min, and hold at 1% B until the 20 min mark. The column compartment was kept at 45 °C and the flow rate was 0.25 mL/minute. The injection volume was 10 µL and the autosampler temperature was 4 °C. The mass spectrometer was operated in full-scan positive and negative ion modes (two separate injections per sample), with spray voltages of +3.5 kV and −3.5 kV, respectively. The scan range was m/z 172.10692 and 170.09126, respectively). Semiquantitative concentrations are expressed as µg metabolite per g cells , using the following settings: precursor mass tolerance: 5 ppm, maximum retention time shift: 30 s, mzCloud match factor threshold: 95%. Compounds without MSMS (identified only using accurate mass) and those without a full mzCloud match were filtered out. Redundant features were collapsed by selecting the single identification with the highest mzCloud match score (including repeated identifications within/between runs and between positive and negative ionization modes). Redundant features with the same mzCloud score were collapsed by selecting the single feature with the highest peak area. Peak areas were normalized using isotopically labeled (13C6) phenylalanine in both positive- and negative-ion modes . The data were tested for normality using the Shapiro–Wilk test and for homoscedasticity using the Brown–Forsythe test. the Relative ATP levels, cell viability, cytotoxicity, ROS, and MMP data were analyzed using a Kruskal–Wallis test followed by a Dunn’s multiple comparison test to determine significance. To simplify comparisons and increase statistical power, comparisons on graph depict those to the medium control only. However, in p < 0.05 -adjusted)). Volcano plots were created comparing selected groups; highlighted metabolites in the upper left and right quadrants represent statistically significant features with FDR-adjusted p-values < 0.05 and a fold change threshold of 2 (log2-fold change < −1 or > 1). For this experiment, the quality control SRM1950 technical triplicates were clustered tightly within the PCA scores plot and the median percent coefficient of variation for these samples for all metabolites was 13.2%, and 9.59% for biopterin  using log-transformed, Pareto-scaled (mean-centered divided by the square root of the standard deviation of each variable), and internal standard-normalized semi-quantitative peak areas using MetaboAnalyst 5.0 software . DiffereWe first tested the effects of increasing concentrations of butyrate and TNFα on cell viability and cytotoxicity in rat primary CECs. Considering the wide range of physiologic concentrations reported in the literature, we aimed to establish the temporal  and dose-dependent interaction between the two agents in rat CEC culture. Based upon these data, we chose to investigate only the 24 h time point for mitochondrial bioenergetics, gene expression analysis, and metabolomics.p < 0.0001) and cytotoxicity  in primary rat CECs with butyrate and TNFα concentrations at 24 h (p < 0.05) (p < 0.05). Co-treatment of 500 µM butyrate over increasing concentrations of TNFα (500 pg/mL–1 ng/mL) did not affect cell viability  A. Butyraiability A. When CTNFα treatment alone increased CEC cytotoxicity , 24 h , and 72 h    . At 4 h, 0.0001) . This wa < 0.05) . These dp = 0.0004), 24 h , and 72 h  (p < 0.05)  . At 4 h, 0.0001) . At 24 h 0.0001)  in MMP w 0.0001) . A diffe < 0.05) . Taken t < 0.05) . = 55.78, p < 0.0001)  = 10.68, p < 0.0001)  = 91.06, p < 0.0001)  , intrace 0.0001) , as well 0.0001) . At 24 h 0.0001)  compared 0.0001)  compared 0.0001) . However 0.0001) , it was p < 0.0001). At 24 h, TNFα alone produced a significant 30% and 90% decrease in ROS at the two highest concentrations only   . Intergr 0.0001) , suggest = 5.52, p = 0.0070)  = 6.21, p = 0.0043)  = 6.10, p = 0.0047)  = 9.19, p = 0.0007)  = 8.37, p = 0.0012)  = 2.90, p = 0.0609)  = 3.45, p = 0.0369)  B, basal  0.0043) C, ATP-li 0.0047) D, maxima 0.0007) E, spare  0.0012) F, and no 0.0609) H, but no 0.0369) G, compar 0.0369) B.  = 10.85, p = 0.0003), basal respiration  = 16.93, p < 0.0001), ATP-linked respiration  = 30.25, p < 0.0001), and maximal respiration  = 5.10, p = 0.0083)  = 1.78, p = 0.1861), proton leak  = 1.90, p = 0.1678), nor non-mitochondrial respiration  = 1.86, p = 0.1712)  C–E but d 0.1712) F–H. Inte 0.1712) C–E. Buty 0.1712) C,D, sugg 0.1712) F–H. = 6.10, p = 0.0041), glycolytic reserve  = 10.32, p = 0.0003), and non-glycolytic acidification  = 4.36, p = 0.0155), but not glycolysis  = 1.83, p = 0.1762) or glycolytic capacity  = 6.211, p = 0.0037) when compared to the control group  = 2.912, p = 0.0524), glycolytic acidification  = 2.676, p = 0.0671), glycolytic capacity  = 3.736, p = 0.0234), glycolytic reserve  = 5.168, p = 0.0068), and non-glycolytic acidification  = 22.79, p < 0.0001)  A–F. As s 0.0001) A,E and i 0.0001) D,F when  0.0001) F when coBased on our mitochondrial and cytotoxicity/cell viability data, we selected 500 µM butyrate and 500 pg/mL TNFα alone and in co-treatment to further probe mechanisms via gene expression and metabolomics. This concentration of TNFα is within the physiological range of that reported for hypertensive models ,25, whil = 6.715, p = 0.0038) and Tnfr2  = 9.044, p = 0.0010) were both decreased in cells treated with TNFα in the presence or absence of butyrate. Compared to the medium control, the transcript levels of the epithelial stem cell marker Lgr5 significantly decreased following TNFα treatment alone  = 5.148, p = 0.0111) but not in any other treatment group. Compared to the medium control, Pdk4 expression levels were upregulated by TNFα and butyrate co-treatment  = 16.77, p < 0.0001) with no changes observed in any other treatment group. The monocarboxylate transporters of the short-chain fatty acids Slc16a1  = 11.37, p = 0.0003) and Slc16a3  = 2.332, p = 0.1128) were downregulated by TNFα treatment, with Slc16a1 expression levels also downregulated by butyrate treatment alone. No significant differences were detected among the groups for transcript levels of Bax, Bcl2, Cat, Sod1, Sod2, Cs, Pdk1, Pdk2, Ppargc1a, and Spr  = 5.640, p = 0.0039)  C. Butyrap < 0.05)  , which m < 0.05) .This study demonstrated the following novel findings: (i) A significant interaction between butyrate and TNFα was observed at some concentration for cell viability, cell cytotoxicity, and calcium ratios were tested in primary rodent CECs, suggesting that butyrate may protect against TNFα-induced changes in these variables. (ii) No significant interactions between butyrate and TNFα were observed when measuring ATP production, MMP, or ROS. However, one or both treatments alone were able to affect said variables, as discussed in detail in the below text. (iii) TNFα significantly decreased several metrics of CEC respiration and energy production, including glycolytic, reserves and non-glycolytic acidification. Butyrate co-treatment did not modify the TNFα-induced effects on these variables in CECs. (iv) Co-treatment of butyrate and TNFα increased Pdk4 expression but decreased glycolysis, OxPhos, and ATP levels, suggesting effects on epithelial lipogenesis. (v) TNFα-induced biopterin levels in colonocytes were augmented, while co-treatment with butyrate was unable to mitigate the effect on biopterin levels. Pdk4 transcripts during TNFα and butyrate co-treatment. Additionally, as early as 3 weeks of age, the SHRs have significantly elevated expression of the catalytic subunit of AMPK (Prkaa2), suggesting a low AMP-to-ATP ratio [Our data indicate that both butyrate and TNFα can induce a quiescent energy phenotype in CECs by lowering ATP levels in a dose-dependent manner. Our gene expression data support this, as we observed increased levels of TP ratio ,35. HoweTP ratio . ConsideTP ratio ,37, the TP ratio , as wellTP ratio , the rolWe first aimed to establish cytotoxic thresholds in primary rat CECs. Our data indicate that micromolar concentrations of butyrate may counteract the cytotoxicity induced by TNFα. Cytotoxicity can be defined as a reduction in metabolic activity with a corresponding decrease in cell membrane potential, whereas cytostatic effects can be described as reductions in metabolic activity without a corresponding decrease in cell membrane potential . Our datButyrate alone was observed to elevate mitochondrial membrane potential (MMP) in a dose-dependent manner, while TNFα treatment alone decreased MMP. Moreover, the presence of butyrate in the cell culture counteracted the TNFα-induced decrease in MMP, suggesting that butyrate can exert protective effects on cell viability by stabilizing MMP. MMP is the electrical potential (ΔΨm) established by the charge separation of protons in the intermembrane space to generate ATP, to import cations such as calcium and iron, and to facilitate the mitophagy of depolarized mitochondria ,42. MMP The detrimental effects of TNFα on MMP may be secondary to increased calcium signaling and ROS production ,46. SincIt is noteworthy that at 24 h, butyrate reduced ROS production with or without TNFα, even in the presence of elevated MMP, which is likely related to the quiescent energy phenotype. Pro-oxidants are causally linked to, whereas antioxidants are protective against, HTN , suggestAlthough we did not investigate them directly, alterations in MMP are also associated with potential changes in iron uptake in the colon. Specifically, the uptake of iron from the cytosolic labile iron pool (CLIP) to mitochondria has been shown to be dependent on MMP ,61. MitoPhysiological concentrations of TNFα exerted an inhibitory effect on Tnfr2 and Casp3 expression at 24 h, both in the presence and absence of butyrate. These data suggest pro-survival action, as the inhibition of caspase-3 has been shown to decrease responsiveness to TNFα-induced apoptosis . AdditioLeucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5) is a stem cell marker essential to colonic homeostasis . Lgr5 ex2 cells by hyperacetylating histones in the promoter region of Pdk4 [Pdk4 transcripts were upregulated when cells were exposed to both butyrate and TNFα. Although we did not measure enzyme activity, PDK4 has been shown to switch the cells away from glycolysis toward fatty acid metabolism [slc16a1 and slc16a3, fatty acid transporters that can shuttle lactate, pyruvate, and SCFAs such as butyrate [Along with the reductions in glycolytic acidification observed following butyrate and TNFα co-treatment, our gene expression data provide further evidence for impaired glucose utilization in colonocytes exposed to TNFα and butyrate. Butyrate is the preferred energy source for differentiated CECs ,71, and  of Pdk4 , increastabolism . Our datbutyrate ,76. In tbutyrate . The dowbutyrate ,77, suggbutyrate . FurtherOur metabolomics data show an increase in biopterin levels following TNFα treatment. Furthermore, butyrate appeared to augment biopterin levels when co-administered with TNFα, despite not significantly increasing biopterin levels when administered alone. Biopterins are enzymatic co-factors essential for the synthesis of amines such as norepinephrine, serotonin, and histamine, as well as trace amines ,80, utilOf note, a transcriptomics dataset of the colonic epithelium of 3-week-old SHR and WKY rodents  shows thMetabolically, biopterins can exert direct effects on isolated mitochondria, acting as an electron transfer cofactor, which, at high concentrations, can inhibit ATP formation by reducing cytochrome oxidase . In lineButyrate, however, appears to be a relatively weak inducer of biopterin, despite its reported role in inducing the production of serotonin, for example, in the gut ; in the Several inflammatory conditions of the colon involving elevated TNFα present with increased ROS production, microbial dysbiosis, and altered circulating butyrate and other SCFA levels or transport. Butyrate regulates the host–microbiota axis, with important implications for inflammation , and the


The prevalence of MDR-GNB isolated from burns patients has increased dramatically in the last decade, representing a serious risk to patients admitted to burns units worldwide. The challenges of managing infected burns patients are exacerbated in poor resource settings. This study was designed to develop a pathway for the rapid diagnosis of multidrug-resistant (MDR) Gram-negative infections and identify the bacterial genes including blaOXA1-blaSHV-blaTEM and carbapenemases genes blaOXA48-blaKPC-blaNDM-blaVIMClinical isolates were collected (August 2017 to August 2018) from Intensive care unit (ICU) of Burn Centre. Antibiotic susceptibility testing and phenotypic detection of ESBLs and carbapenemases was performed by disk diffusion, double disk synergy test (DDST), combination disk test (CDT), and Imipenem + EDTA combined disk test (IMP + EDTA CDT). Polymerase chain reaction (PCR) detection was performed for ESBLs Pseudomonas aeruginosa was found to be the most prevalent 43/104 (41.4%), followed by Klebsiella pneumoniae 17/104 (16.4%), Acinetobacter baumannii12/104 (11.5%), and 6/104 Proteus mirabilis (5.8%). All isolates (100%) were resistant to cefotaxime and ceftazidime, while the meropenem resistance was 58.7%. ESBL and carbapenemase genotypes were found to be associated with higher MAR index (0.65–0.88) and MIC (> 32 µg/ml) values P. aeruginosa was the major ESBL and carbapenemase producer as determined by phenotypic testing and PCR. blaTEM positive isolates among ESBLs producers were predominant 81.8% (27/33), followed by 27.3% blaOXA1 and blaSHV, respectively. blaVIM positive isolates among carbapenemase producers were predominant 47.7% (21/44), followed by 27.3% blaKPC, 20.5% blaOXA48, and 11.4% blaNDM positive isolates.In total, of 170 Gram-negative isolates, 104 (61.2%) were confirmed as multidrug-resistant (MDR); Pseudomonas aeruginosa. There are only limited effective antibiotics against such strains. blaVIM and blaTEM individually and in co-existence with blaKPC, blaOXA48, blaSHV, and blaOXA1 confer antimicrobial resistance in burns patients. Rapid detection of ESBL and carbapenemase genes will inform treatment strategies improving the outcome for post-burn patients in ICU.The predominant organism causing burn infections was ESBL and carbapenemase-producing  Pseudomonas aeruginosa is a common infection in burns patients, as are Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis [Klebsiella pneumoniae are distributed in Australia, China, Central and South America. In contrast, the TEM type ESBLs persist in France and North America, and Africa [Pseudomonas aeruginosa against oxacillin and cephalosporins have been reported from India and Iran. However, very little is known about their worldwide distribution [blaKPC, blaNDM, blaVIM, and blaOXA48 can be detected in MDR-GNB [K. pneumoniae, and P. aeruginosa strains from the United States [Enterobacteriaceae and Acinetobacter baumannii strains [irabilis , 10. Thed Africa , 12. OXAribution , 14. Gen MDR-GNB . KPC andd States . NDM car strains , 18. OXA strains , 19. ThiblaOXA1-blaSHV-blaTEM and carbapenemases by blaOXA48-blaKPC-blaNDM-blaVIM multiplex PCR [A cross-sectional study was conducted at Jinnah Burns and Reconstructive Surgery Centre (JB&RSC)/Allama Iqbal Medical College (AIMC), Lahore, Pakistan and the Department of Microbiology and Molecular Genetics, University of the Punjab, Pakistan, in collaboration with UCL Centre for Clinical Microbiology, London, United Kingdom. The burns unit consists of 75 beds, and the clinical specimens were collected between August 2017 and August 2018 from 170 patients being treated in the intensive care unit (ICU). The AIMC Ethics Committee approved the study after the submission of the preliminary proposal (ERB-AIMC 40:12 2017). Patients suffering from previous infections receiving any type of antibiotic therapy were excluded. Specimens including wound swabs, blood, sputum, tracheal aspirates, and urine were collected according to AIMS Standard Operating Procedures (AIMS), 2017, from the patients under treatment in the ICU of Burns Center. Specimen enrichment was performed, and subcultures were carried out on Blood, Chocolate, and MacConkey’s agar plates (Oxoid UK). The identification of bacterial isolates was performed using API-20E and 20NE (Biomerieux France) Cephalosporin and carbapenem-resistant Gram-negative isolates were further processed for phenotypic tests and genetic profiling of ESBLs by Antimicrobial resistance and susceptibility patterns were analyzed by performing Kirby Bauer’s disk diffusion method, and evaluation of MDR  according to Clinical Laboratory Standards Institute  break points. Antimicrobial discs  including piperacillin (PIP 100 µg), amoxicillin-clavulanate (AMC 30 µg), piperacillin-tazobactam (TZP 100/10µg), cefepime (FEP 30 µg), ceftazidime (CAZ 30 µg), cefotaxime (CTX 30 µg), doripenem (DOR 10 µg), imipenem (IMP 10 µg), meropenem (MEM 10 µg), amikacin (AK 30 µg), gentamicin (CN 10 µg), tobramycin (TOB 10 µg), ciprofloxacin (CIP 5 µg), levofloxacin (LEV 5 µg), aztreonam (ATM 30 µg), tetracycline and (TE 30 µg) were used for AST profiling of Gram-negative bacterial isolates. Minimum inhibitory concentrations (MICs) were determined for ceftazidime, cefotaxime, imipenem, meropenem. MDR was defined as acquired non-susceptibility to at least one agent in three or more antimicrobial categories.Phenotypic tests, including double-disc synergism test (DDST) and confirmatory combination disc test (CDT) were performed for ESBLs. Double disk synergism for amoxicillin-clavulanate was tested with cefotaxime, ceftazidime, cephradine, aztreonam, cefuroxime, and ceftriaxone. Cefotaxime and ceftazidime disks with and without clavulanic acid were applied for the confirmation of ESBLs. Carbapenemase detection involved IMP + EDTA combined disk test using the combination of imipenem with ethylene-diamide-tetra acetic acid (EDTA) compared to the only imipenem .Molecular detection of ESBLsblaOXA1-blaSHV-blaTEMand carbapenemasesblaOXA48-blaKPC-blaNDM-blaVIM.blaOXA1-blaSHV-blaTEMPCR included buffer 2.5 µl, MgCl2 1.8 µl, dNTPs 0.6 µl, each primer pair 1.2 µl, Taq-polymerase 0.3 µl for a final volume of 25 µl of reaction. PCR 40 cycles with denaturation at 95 °C for 60 s, annealing at 56 °C for 90 s, and extension at 72 °C 60 s. Qiagen PCR ingredients for blaOXA48-blaKPC-blaNDM-blaVIMPCR included master-mix 10.5 µl, each primer pair 1.5 µl for the final volume of 25 µl of reaction. PCR 40 cycles with denaturation at 95 °C for 30 s, annealing at 50 °C for 30 s, and extension at 72 °C 60 s [https://www.ncbi.nlm.nih.gov/). PCR amplicons from both assays were visualized by agarose gel electrophoresis with 1% agarose gel and 1X Tris-borate-EDTA (TBE) buffer.DNA extraction was performed by the boiling lysis of a cell suspension of pure bacterial colonies. Previously designed conserved region specific primers OXA-1, SHV and TEM for ESBLs and OXA-48 , KPC(F-5 °C 60 s . PCR accPseudomonas aeruginosa 43/104(41.4%) was found to be the most prevalent infectious isolate, followed by Klebsiella pneumoniae 17/104 (16.4%), Acinetobacter baumannii12/104(11.5%), and 6/104 Proteus mirabilis (5.8%). Antimicrobial sensitivity testing confirmed 100% resistance against aztreonam, cefotaxime, ceftazidime, and amikacin. Piperacillin and cefepime resistance were observed in 96.2% and 92.3% isolates, respectively. Resistance against carbapenems was lower; meropenem (58.7%), imipenem (57.7%), and doripenem (56.7%). Colistin resistance was observed in 5.8% isolates , and n = 104 were MDR Gram-negative bacterial pathogens of post-burn infections in ICU patients. P. aeruginosa, K. pneumoniae and P. mirabilis, positive for both the ESBLs and carbapenemases. blaTEM positive isolates were predominant at 81.8% (27/33), followed by 27.3% blaOXA1 and blaSHV, respectively. blaSHV-blaTEM co-existence was observed in 21.2%, followed by blaOXA1- blaTEM in 9.1% and blaOXA1-blaSHV-blaTEM in 3% isolates , whereas genotyping by multiplex PCR yielded 31.7% (33/104) positive isolates. Carbapenemase detection by IMP-EDTA combination disk test resulted in 52.9% (55/104), and multiplex PCR yielded 42.3% (44/104) positive isolates  followed by 27.3% blaKPC, 20.5% blaOXA48, and 11.4% blaNDM positive isolates. blaKPC-blaVIM and blaOXA48-blaKPC-blaVIM co-existence was observed in P. mirabilis and E. cloacae, respectively  bacterial pathogens . The cureviously , 4. Theseviously , 15. Theeviously . Burns peviously . The higeviously , 18.P. aeruginosa, and VIM-like carbapenemases, which agrees with studies reported from Algeria and China [A. baumannii isolated in Iran [Enterobacteriaceae have been frequently reported from India, Pakistan, and China [E. coli strains [A. baumannii isolates from burn patients in Iraq with more than 70% resistance against cephalosporins and carbapenems [We have observed that isolates from wounds are mostly associated with nd China , 25. OXA in Iran . NDM-prond China , 26. Sur strains . TEM folbapenems .There are minimal data on antimicrobial resistance and molecular profiling of ESBLs and carbapenemases from burns patients in Pakistan. The co-existence of more than one genetic variant encoding ESBLs produces a masking effect decreasing the diffusion or permeability of the antibiotic, leading to false-negative results , 11. ThePseudomonas aeruginosa is the frequently ESBLs and carbapenemase-producing strain isolated from burns patients. VIM carbapenemases, TEM ESBLs individually and in co-existence with KPC, OXA-48, and SHV and OXA-1 ESBLs confer antimicrobial resistance in burns patients. Here we report the development of a pragmatic diagnostic strategy for ESBLs and carbapenemase-producing clinical isolates which provides a presumptive diagnosis to inform rapidly the selection of antibiotic therapy: an exceptional diagnostic and clinical strategy is required to combat such an alarming situation to improve healthcare and control the spread of infections. Therefore, it is necessary to properly manage phenotypic and molecular methods to provide complete resistance profiles to ensure appropriate antibiotic administration.In conclusion, the rates of ESBLs and carbapenemases producing strains among ICU burns patients is high. In our setting