In transplantation groups, perfusate glucose consumption was higher in the control group. Lactate levels were significantly lower in the PFCOC group (p = 0.02). Perfusate flavin mononucleotide (FMN) was significantly higher in the controls (p = 0.008). Post-transplant gas exchange was significantly better during the 4-h reperfusion period in the PFCOC group (p = 0.01). Plasma IL-8 and IL-12 levels were significantly lower in the PFCOC group . ATP lung tissue levels at the end of the transplantation were higher and myeloperoxidase (MPO) levels in lung tissue were lower in the PFCOC group compared to the control group. In the PFCOC group, TEM showed better tissue preservation and cellular viability. Conclusion: PFCOC application is safe during EVLP in lungs preserved 24 h at 4 °C. Although this strategy did not significantly affect the EVLP physiology, metabolic markers of the donor quality such as lactate production, glucose consumption, neutrophil infiltration and preservation of mitochondrial function were better in the PFCOC group. Following transplantation, PFCOC resulted in better graft function and TEM showed better tissue preservation, cellular viability and improved gas transport.Background: Ex vivo lung perfusion (EVLP) is a technology that allows the re-evaluation of questionable donor lung before implantation and it has the potential to repair injured donor lungs that are otherwise unsuitable for transplantation. We hypothesized that perfluorocarbon-based oxygen carrier, a novel reconditioning strategy instilled during EVLP would improve graft function. Methods: We utilized perfluorocarbon-based oxygen carrier (PFCOC) during EVLP to recondition and improve lung graft function in a pig model of EVLP and lung transplantation. Lungs were retrieved and stored for 24 h at 4 °C. EVLP was done for 6 h with or without PFCOC. In the transplantation groups, left lung transplantation was done after EVLP with or without PFCOC. Allograft function was assessed by means of pulmonary gas exchange, lung mechanics and vascular pressures, histology and transmission electron microscopy (TEM). Results: In the EVLP only groups, physiological and biochemical markers during the 6-h perfusion period were comparable. However, perfusate lactate potassium levels were lower and ATP levels were higher in the PFCOC group. Radiologic assessment revealed significantly more lung infiltrates in the controls than in the PFCOC group ( Due to graft shortage, approximately 10–13% of lung transplant candidates die while on the waiting list every year ,3 AdditiEx vivo lung perfusion (EVLP) has become an alternative to conventional approaches for donor lung assessment, reconditioning, and preservation in clinical lung transplantation .EVLP is a technology that allows the re-evaluation of questionable donor lung before implantation and it has the potential to repair injured donor lungs that are otherwise not suitable for transplantation ,14,15. Normothermic ex vivo organ perfusion was reported as early as 1935 . Steen e2) carrying blood substitutes has evolved from replicating blood O2 transports properties to that of preserving microvascular and organ function, reducing the inherent or potential toxicity of the material used to carry O2, and treating pathologies initiated by anemia and hypoxia. Furthermore, the emphasis has shifted from blood replacement fluid to so-called “O2 therapeutics” that restore tissue oxygenation to specific tissues regions [2 and other gases [2 is released, carbon dioxide (CO2) is taken up. Oxygen dissolved in PFC is immediately available to tissues with a high extraction ratio [The goal of developing oxygen  during EVLP to recondition and improve lung graft function in a large animal model of EVLP and lung transplantation.All animals received humane care during the experiments in accordance with the updated “The Guide for the Care and Use of Laboratory Animals” . The Canton of Zurich Veterinary Authorities approved the animal use protocol (ZH 150/16).(1)(i)PFCOC: Lungs were retrieved and stored for 24 h at 4 °C. PFCOC emulsion was added to the EVLP circuit (n = 7). EVLP time was 6 h.(ii)Control: Lungs were retrieved and stored for 24 h at 4 °C. EVLP was performed without PFCOC (n = 6). EVLP time was 6 h.EVLP Only Groups(2)(i)Control: Lungs were retrieved and stored for 24 h at 4 °C. EVLP was performed without PFCOC. The left lung was transplanted after 6 h of EVLP. Observation of the transplanted lung was 4 h (n = 5).(ii)PFCOC: Lungs were retrieved and stored for 24 h at 4 °C. PFCOC emulsion was added to the EVLP. The left lung was transplanted after 6 h of EVLP. Observation of the transplanted lung was 4 h (n = 4).(iii)Immediate Transplantation (Im-Tx): Following 4 h of cold ischemic storage, the left lung was transplanted. Observation of the transplanted lung was 4 h (n = 3).Transplantation GroupsDomestic female pigs (25–35 kg) were used for all study phases. Pig lungs were randomly assigned into study groups. Donor lungs retrieved using standard protocol and preserved at 4 °C for 24 h with Perfadex  followed by EVLP according to a previous report . Lungs wIn transplantation groups, the recipient operation was started after 5 h of EVLP. After thoracotomy, the right pulmonary artery and right main bronchus were encircled followed by a left pneumonectomy. Orthotopic left single lung transplantation was then performed and the recipients were observed for 4 h.The allograft function was assessed hourly during the 6 h EVLP and after reperfusion by means of pulmonary gas exchange, lung mechanics and vascular pressures. In the transplantation groups, at the end of 4 h of reperfusion, the allograft was challenged with a contralateral pulmonary artery occlusion for 5 min, followed by clamping of the right main bronchus for evaluation of the isolated allograft mechanics.Perfusate and plasma samples were collected hourly. The Epoc blood analysis system  was used to measure perfusate and blood gases and for clinical ions biochemistry.At the end of EVLP and transplantation, a bronchoalveolar lavage was performed with 20 mL of cold saline from the lower lobe segments of the right and left lungs, respectively. Approximately 15 mL were recovered during each wash. The recovered wash fluid was subjected to cytological assessment at the hospital’s laboratories, using May–Grünwald–Giemsa stained cytological specimens and were subsequently processed as previously described for further analysis . SimilarThe inflammatory response in perfusate, plasma, and BAL samples were profiled using a 13-plex porcine cytokine/chemokine array . Lung tissues were collected and flash frozen in liquid nitrogen and then stored at −80 °C. Powdered frozen lung tissue (25 mg) were homogenized in 0.5 mL of 0.5% trichloroacetic acid and centrifuged at 8000 rpm for 2 min at 4 °C. The supernatant was isolated and 10× concentrated Tris-acetate buffer was used to neutralize pH to 7.4 with a 10 mL of 0.002% xylenol blue used as the pH indicator. Myeloperoxidase (MPO) activity was measured using a kit (BioVision Inc cat K744-100) according to the manufacturer’s instructions, and the results were expressed in milliunits per milliliter.We used an ATP assay kit  to enzymatically estimate the ATP concentration in the supernatant by measuring it in the luminescence channel of a Cytation 5 plate reader . Lung tissues were collected and flash frozen in liquid nitrogen and then stored at −80 °C. Frozen lung tissues (25 mg) were homogenized in 0.5 mL of 0.5% trichloroacetic acid and centrifuged at 8000 rpm for 2 min at 4 °C. The supernatant was isolated and 10× concentrated Tris-acetate buffer was used to neutralize the pH to 7.4 with 10 mL of 0.002% xylenol blue used as the pH indicator. A microBCA protein assay kit was used with bovine serum albumin as standard to determine protein concentration in the supernatant of each sample tested according to the manufacturer’s instructions .The microplate was analyzed by fluorescence spectroscopy. A monochrome light with an excitation wavelength of 485 nm was used and the fluorescence with a wavelength of 528 nm emitted by the FMN was recorded with 100% gain. The fluorescence readings in the artificial unit (AU) were displayed in an Excel spreadsheet produced by the spectrometer. This experiment was performed 3 times .At the end of EVLP a radiologist evaluated the lung X-rays in a blinded fashion as previously described .Tissue samples taken at the end of EVLP and transplantation were stained with hematoxylin and eosin and blindly assessed by two veterinary pathologists for any pathological changes. The most prominent features observed in the transplanted lungs were used to develop a scoring system which was as follows: (A) Evidence of neutrophil recruitment into the lungs, represented by the presence of neutrophils within vessels ; (B) Evidence of neutrophil emigration into the tissue ; and (C) Evidence of neutrophils within the tissue, represented by the presence of individual neutrophils within alveolar lumen .Transmission electron tissue samples were fixed in 2.5% glutaraldehyde (EMS) buffered in 0.1 M Na-P-buffer overnight, washed ×3 in 0.1 M buffer, post-fixed in 1% osmium tetroxide (Sigma-Aldrich) and dehydrated in ascending concentrations of ethanol followed by propylene oxide and infiltration in 30% and 50% Epon (Sigma-Aldrich).At least three 0.9 μm toluidine blue stained semithin sections per localization were produced. Representative areas were trimmed and 90 nm lead citrate (Merck) and uranyl acetate (Merck) contrasted ultrathin sections were produced and subsequently viewed under Phillips CM10, operating with a Gatan Orius Sc1000 (832) digital camera, Gatan Microscopical Suite, Digital Micrograph, Version 230.540.6F13C10H21 (F6H10) (2.8% weight/volume). FnHm diblocks help to (i) control the emulsion droplets’ mean diameter, (ii) strongly stabilize the emulsion (no significant difference in droplet size was detected after one year at 25 °C), and (iii) provide well-tolerated emulsions that were demonstrated to be efficient [Perfluorooctylbromide (PFOB) was distilled twice and detoxified on an alumina column, followed by filtration on a 0.22 µm Millipore filter prior to use. Egg yolk phospholipids were used as the main emulsifier (4.5% weight/volume). The advanced PFOB nanoemulsion (90% PFOB weight/volume) was stabilized using the fluorocarbon/hydrocarbon diblock Cfficient ,48,49,50When we reached perfusion temperature of 37 °C, PFCOC nanoemulsion (0.1 g/kg of FBOB) was injected into the EVLP circuits in a final volume of 20 mL Steen solution via the pulmonary artery line.p-value is less than 0.05.Statistical analyses were performed with PRISM 8 software . All results are expressed as mean ± standard deviation (SD). The Mann–Whitney test was utilized where data were non-continuous. Two-way analysis of variance (ANOVA) for repeated measures was utilized where such data contained a time component. In the transplantation groups, the immediate transplantation (Im-Tx) group was not compared with the other two groups. Differences are considered significant when the 2/FiO2 ratio (control 65.8 ± 3.7 kPa versus PFCOC 60.7 ± 5.6 kPa), donor lung compliance (control 23.22 ± 2.51 mL/mbar versus PFCOC 24.56 ± 4.64 mL/mbar) and cold ischemic time (control 24.19 ± 0.24 h versus PFCOC 24.07 ± 0.11 h) did not differ significantly between the two groups. Only perfusate consumption was significantly lower in the PFCOC group (control 538.3 ± 146.2 mL versus PFCOC 365 ± 97 mL) (p = 0.0455).Donor body weight (control 29.80 ± 3.63 kg versus PFCOC 30.29 ± 4.07 kg), donor PaO2 (control 36.48 ± 3.84 kPa versus PFCOC 44.13 ± 6.65 kPa) (p = 0.188), pulmonary vascular resistance (control 9.95 ± 1.91 Wood units versus PFCOC 9.42 ± 1.65 Wood units) (p = 0.7) and lung compliance (control 15.9 ± 1.65 mL/mbar versus PFCOC 15.29 ± 2.93 mL/mbar) (p = 0.8) were comparable in the two experimental groups.Lung physiology and metabolism were monitored during the 6 h EVLP period. Delta POp = 0.18).Perfusate electrolytes, including potassium and sodium were gradually increased in both groups throughout the EVLP. However, potassium ion concentrations tended to be lower in the PFCOC group than in the control group (control 4.70 ± 0.25 mmol/L versus PFCOC 4.45 ± 0.13 mmol/L) (p = 0.52). Furthermore, lactate levels also increased gradually in both groups throughout the perfusion. However, lactate levels tended to decrease in the PFCOC group compared to the control group, without statistical significance (control 4.89 ± 2.53 mmol/L versus PFCOC 4.23 ± 2.26 mmol/L) (p = 0.12).Perfusate glucose levels were constantly decreased in both groups (control 145.2 ± 17.66 mg/dL versus PFCOC 148.4 ± 17.49 mg/dL) (p = 0.18) and macrophages  but there were more neutrophils  and lymphocytes  in the PFCOC group.In bronchoalveolar lavage (BAL) cell count and differential count at the end of 6 h EVLP, we observed a trend towards a reduction in the total cell count .ATP lung tissue levels tended to be higher in the PFCOC group compared to the control group. However, the difference was not statistically significant (control 844.9 ± 1694 nmol/mg protein versus PFCOC 1415 ± 1125 nmol/mg protein) (p = 0.12).Myeloperoxidase (MPO) activity in lung tissue at the end of 6 h EVLP was lower in the PFCOC group (3.55 ± 2.4 mU/mL) compared to the control group (7.49 ± 2.4 mU/mL) without reaching statistical significance (p = 0.0495)  ( 0.0495) .2/FiO2 ratio (control 65.89 ± 3.41 kPa versus PFCOC 60 ± 6.68 kPa), donor lung compliance (control 23.66 ± 2.53 mL/mbar versus PFCOC 24.85 ± 4.75 mL/mbar), cold ischemic time (control 24.13 ± 0.21 h versus PFCOC 24.1 ± 0.14 h) and perfusate consumption did not differ significantly between the groups (control 536 ± 163.3 mL versus PFCOC 351.3 ± 104.9 mL) (p = 0.127).Donor characteristics including donor body weight (control 29.80 ± 3.63 kg versus PFCOC 31.5 ± 4.65 kg), donor PaOLung physiology and metabolism were monitored during the 6 h EVLP period.2 (control 35.29 ± 3.38 kPa versus PFCOC 48.49 ± 7.63 kPa) (p = 0.1), lung dynamic compliance (control 16.37 ± 1.82 mL/mbar versus PFCOC 15.25 ± 4.21 mL/mbar) (p = 0.7), and pulmonary vascular resistance (control 9.93 ± 1.73 Wood units versus PFCOC 9.60 ± 1.91 Wood units) (p = 0.8) were comparable between groups (Delta POn groups .p = 0.17).Perfusate electrolytes, including potassium and sodium increased gradually in both groups throughout the EVLP. However, potassium ion concentrations tended to be lower in the PFCOC group than in the control group (control 4.70 ± 0.25 mmol/L versus PFCOC 4.32 ± 0.08 mmol/L) (p = 0.3). Furthermore, lactate levels also increased gradually in both groups throughout the perfusions. However, lactate levels were significantly lower in the PFCOC group (control 4.91 ± 2.55 mmol/L versus PFCOC 3.73 ± 2.07 mmol/L) (p = 0.0249) (Perfusate glucose levels were constantly decreased in both groups (control 144.5 ± 18.12 mg/dL versus PFCOC 149.7 ± 16.21 mg/dL) ( 0.0249) .p = 0.11) and macrophages  but there were more neutrophils  and lymphocytes  in the PFCOC group.In BAL, we observed a trend towards fewer total cells . The systemic arterial gas exchange (control 61.03 ± 4.57 kPa versus PFCOC 65.89 ± 9.08 kPa), allograft pulmonary graft venous oxygen concentration (control 55.06 ± 1.59 kPa versus PFCOC 73.72 ± 4.45 kPa), and dynamic compliance (control 19.54 ± 3.05 mL/mbar versus PFCOC 22.89 ± 2.04 mL/mbar) were comparable between the control and treated group.Hemodynamic and physiologic parameters during the 4 h of reperfusion are shown in p = 0.0159) (p = 0.2)  ( 0.0159) . When wep = 0.2) .p = 0.01, and p = 0.03, respectively).p = 0.11), IL-1β , IL-1Ra , and IL-12  were markedly reduced in the PFCOC group compared to the control .ATP lung tissue levels at the end of the transplantation (after 4 h of reperfusion) were numerically higher in the PFCOC group compared to the control group . In the immediate transplant group MPO lung tissue levels were 0.01 ± 0.01 mU/mL.MPO levels in lung tissue after 4 h reperfusion in the transplant groups tended to be lower in the PFCOC group (4.89 ± 2.5 mU/mL) compared to the control group , respectively (The level of flavin mononucleotide (FMN) in the EVLP perfusate was significantly higher in the control group compared to PFCOC group: time 0 (baseline): 5489 ± 607 versus 5327 ± 458; EVLP 1 h: 5309 ± 247 versus 4881 ± 412; EVLP 2 h: 5805 ± 326 versus 5282 ± 570; and EVLP 3 h: 6227 ± 221 versus 5827 ± 351  are a class of chemicals that are essentially composed of carbon and fluorine atoms ,38,39,52PFC-based emulsions have the capacity to dissolve large concentrations of oxygen, they can be produced with small particle sizes, and they are low-viscosity suspensions ,54,55,56The anti-inflammatory effects of PFCs either by direct modulation of inflammatory cell function or attenuation of the production of inflammatory mediators have been reported ,42,43,4440. This effect was explained by the inhibition of NF-kB activity, inflammatory reactions and neutrophil infiltration into the lung tissue [Chu et al. used an isolated rat lung perfusion model to demonstrate the beneficial effects of systemic PFC administration on ischemia reperfusion injury g tissue . In isolg tissue . Schroedg tissue . The smag tissue . In rat g tissue . PFCs reg tissue ,65,66. Pg tissue ,68 and rg tissue . A PFOB g tissue . Adminisg tissue . Resuscig tissue . Acute lg tissue . A PFOB g tissue .In a phase 3 study, PFC decreased allogeneic erythrocyte transfusions in major non-cardiac surgical procedures associated with significant blood loss ,75. In eAs PFCs are not metabolized, but excreted unchanged via the lungs, their potential for cytotoxicity is thought to be limited . There iIn our study, the control group exhibited mild early inflammatory reaction (activated endothelium and intravascular neutrophils) and evidence of neutrophil emigration . Lower MIntravenous infusion of perfluorocarbon, did not cause additional vasoconstriction in cerebral pial arterioles or increase systemic blood pressure compared with saline and colloid in healthy anesthetized rat cranial window method . This isUse of PFCOC emulsion prevented the adhesion of established pancreatic beta cell lines and the oxygen-carrying capacity of PFCOC emulsion may also help to prevent the period of hypoxia exposure, which is known to contribute to the failure of post-transplant survival . GlucoseIn accordance to the above-mentioned experimental and clinical observations in our study, perfusate glucose levels decreased constantly in both groups, but the control group showed a trend toward more glucose than the PFCOC group. In addition, lactate levels also increased gradually in both groups throughout the perfusion. However, lactate levels were lower in the PFCOC group compared to the control group.Flavin mononucleotide (FMN) is a small molecule bound to the mitochondrial complex I . The losIn our study, following lung transplantation, the lungs principally exhibited the same morphological features. There was some degeneration and cell death, and we observed the presence of intracapillary macrophages  and macrophages that form small clusters in the interstitial space.The immediate transplant group had a higher number of macrophages in the alveolar septa compared to the control and treated groups, which might be a consequence of EVLP flushing. The macrophages were shown to proliferate. These features are also seen in EVLP groups only. Transplantation is associated with an acute inflammatory reaction, represented by endothelial cell activation, neutrophil rolling and emigration. The extent of this inflammatory response appears to be lowest in the immediate transplantation group. However, there was a tendency for a less intense inflammatory reaction in PFCOC group.TEM showed another interesting feature, that is, there was distinct vesiculation of capillary endothelial cells and to a lesser extent, vesiculation in type I pneumocytes in the PFCOC group. We interpreted this finding as evidence of trans-membranous transport or gas exchange. It appears to be attributable to PFCOC treatment, since otherwise endothelial cell vesiculation was only seen in normal lung and in the treated group.In conclusion, perfluorocarbon-based oxygen carrier (PFCOC) application is safe during EVLP in lungs preserved 24 h at 4 °C. Although this strategy did not significantly affect the EVLP physiology, metabolic markers of donor quality such as lactate production, glucose consumption and preservation of mitochondrial function were better in the treatment group. Inflammation was lower in the treatment group. Moreover, perfusate enrichment improves electrolyte imbalance to a certain degree during EVLP. Following transplantation, PFCOC provided better graft function and TEM showed better tissue preservation and cellular viability compared to controls.


Gcg expression and reduced glucagon-like peptide-1 (GLP-1) secretion in vitro. In mice, administration of p-cresol in the drinking water for 2 weeks reduced the transcript levels of Gcg and other gut hormones in the colon; however, it did not affect either fasting or glucose-induced plasma GLP-1 levels. Furthermore, it did not affect glucose tolerance but promoted faster small intestinal transit in mice. Overall, our data suggest that microbial metabolite p-cresol suppresses transcript levels of gut hormones and regulates small intestinal transit in mice.p-cresol is a metabolite produced by microbial metabolism of aromatic amino acid tyrosine. p-cresol and its conjugated forms, p-cresyl sulfate and p-cresyl glucuronide, are uremic toxins that correlate positively with chronic kidney disease and diabetes pathogenesis. However, how p-cresol affects gut hormones is unclear. Here, we expose immortalized GLUTag cells to increasing concentrations of p-cresol and found that p-cresol inhibited  Gut microbiota regulates the production of a plethora of microbial metabolites that are recently revealed to play an important role in the host’s metabolic regulation , 2. Fermvia their action on peripheral organs such as the pancreas influencing glucose metabolism (Enteroendocrine cells (EECs) reside along the length of the intestine and sense fluctuations in dietary and gut microbiota-regulated metabolites . Multipltabolism , 19. It tabolism . Therefoin vitro and in vivo procedures to unravel the effects of p-cresol on gut hormone expression and secretion. We found that p-cresol inhibits proglucagon (Gcg) expression in vitro and that p-cresol administration to mice reduced the RNA expression of Gcg and other gut hormones in the colon and decreased small intestinal transit time in mice.To this end, we used both GLUTag cells were seeded at the density of 100,000 cells per well in poly-D-lysine-coated 24-well plates. All experiments were performed 24–48 h after seeding when cells reached 70%–80% confluence. For the expression assay, cells were washed once with Dulbecco’s Modified Eagle’s Medium (DMEM) containing 1% fetal bovine serum (FBS) and 0.2% bovine serum albumin (BSA) and incubated with different concentrations of p-cresol  for 24 h. The cells were scraped and collected in RLT lysis buffer containing 10 µl β-mercaptoethanol/ml. The lysates were snap-frozen until RNA extraction.3, 1.2 mM NaH2PO4, 2.6 mM CaCl2, 1.2 mM MgCl2, and 10 mM HEPES, pH 7.4) containing 0.1% BSA. The cells were then incubated with different concentrations of p-cresol  with or without positive control, 10 µM chemical TGR5 agonist MerckV, for 2 h. Following incubation, secretion buffer was collected and centrifuged at 1,500×g to remove any floating cells. Finally, supernatants were collected and snap-frozen until GLP-1 analysis. All secretion and expression assays were repeated independently at least three times, and statistics was performed on the average of data points per independent experiment.For the secretion assay, the cells were washed three times with secretion buffer  for 2 weeks. Water bottles containing p-cresol were changed twice weekly. Body weight, food and water intake were monitored two times a week. At the end of the experiment, blood samples were collected by puncturing the retro-orbital sinus and plasma was separated by centrifugation at 10,000×g for 5 min. Liver, ileum, cecum, and proximal colon were collected and snap-frozen. In a separate cohort of female mice (n = 4–5), following 2 weeks of p-cresol supplementation, an oral glucose tolerance test was performed by administering 30% glucose solution (2 g/kg body weight) to 4-h fasted mice and blood samples were collected from the retro-orbital sinus at baseline and 10 min following glucose gavage. Glucose readings were taken before and 10, 30, 60, 90, and 120 min after the oral glucose gavage. Three days later, 4-h fasted mice were orally gavaged with 200 µl of 6% carmine red solution prepared in 0.5% methylcellulose to assess small intestinal transit. After 45 min, whole small intestinal tract was removed and a visible proportion (by eye) of small intestine in which carmine red has traveled was expressed as the percentage of total small intestinal length to determine small intestinal transit as described before  accepted principles for the care and use of laboratory animals approved by the Animal Experiments Inspectorate under the Danish Ministry of Food, Agriculture and Fisheries. Swiss Webster males (n = 6–7) were group-housed in individual ventilated cages at a temperature- and humidity-controlled room at 22°C with 12:12 light/dark cycle. Mice were fed a Western-style diet  and then randomly allocated to receive either drinking water or water supplemented with 0.25 g/L p-cresol  was used as endogenous control, whereas for cDNA from mouse tissue, the housekeeping gene Ywhaz (tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta) was used as endogenous control. Relative expression was calculated using 2-ΔΔCt method. Forward and reverse primer sequences are as follows: Gapdh 5´-AGGGCTCATGACCACAGTC-3´, 5´-GGATGCAGGGATGATGTTCT-3´; Ywhaz 5’-AGACGGAAGGTGCTGAGAAA-3’, 5’-GAAGCATTGGGGATCAAGAA-3’; Gcg 5´-GCTTATAATGCTGGTGCAAG-3´, 5´-TTCATCTCATCAGGGTCCTC-3´; Pyy 5´-GCAGCGGTATGGAAAAAGAG-3´, 5´-GTCGCTGTCGTCTGTGAAGA-3´; Nts 5´-CTGGTGTGCCTGACTCTCCT-3´, 5´-TCACATCTTCTTCTGAATCTGAGC-3´; Cck 5´-CCCCAATGTGAAATCTGTCC-3´, 5´-GGTCTGGGAGTCACTGAAGG-3´.RNA was extracted from cell lysates and intestinal segments following kit instructions . cDNA was synthesized using the SuperScriptTM III Reverse Transcriptase (ThermoFisher Scientific). Gene expression was analyzed in a qPCR reaction containing 5 μl PrecisionPLUS 2× qPCR Master Mix (Primer design), 0.5 μl forward/reverse primer (10 μM), and 2.5 μl sterile water per reaction. For cDNA from GLUTag cell experiments, the housekeeping gene Total GLP-1 was analyzed in the secretion buffer from GLUTag cells and plasma samples from mice using V-plex GLP-1 Total kit on the Mesoscale Discovery platform.Method applied was previously reported  and was In the first step, tissue samples were cryo-pulverized using CPO2 . Deep frozen tissue was inserted into cryo-bag  and pulverized. Powder was stored on -80°C until further processing. Water was removed from the samples with ScanVac CoolSafe freeze drier . Samples were dried for 20 h, and weight was measured. Proteins were removed using organic solvent extraction. Dried tissue powder was mixed with 180 μl of methanol containing 0.1 mg/l of internal standard 3-hydroxy tridecanoic acid and left for 30 min to precipitate proteins on ice. After that, extracts were centrifuged on 4°C and 10,000 rpm for 3 min . Then, 120 μl of supernatant was collected and dried under a stream of nitrogen and resuspended in 60 μl of methanol:water = 1:1 (v:v).A similar procedure was applied for plasma metabolite extraction. Here, 30 µl of plasma was mixed with 180 μl of methanol containing 0.1 mg/l of 3-hydroxy tridecanoic acid. After protein precipitation and centrifugation with the same conditions as tissue samples, 120 µl of supernatant was taken and resolved in 60 µl of methanol:water = 1:1 (v:v). Peak areas were normalized to the internal standard.The system used was composed of high-performance liquid chromatograph  and mass spectrometer . Metabolite separation was achieved with reversed-phase column  and with the gradient of mobile phases. Mobile phase A was water with 0.1% of formic acid, while mobile phase B consisted of acetonitrile:isopropanol = 3:1 (v:v) with added 0.1 % of formic acid. Chromatographic gradient started with 3% of mobile phase B, which was increased to 100% in 9 min where it was kept stable for 5 min. After that system was reequilibrated to initial conditions, column temperature was kept on 40°C and injection volume was 10 μl.Acquisition of mass spectra was set to profiling mode, ranging from 50 to 500 Dalton, and acquisition speed was set to 2 Hz. Spectra were acquired in negative ionization mode. A similar method with automatic acquisition of fragmentation spectra was applied for metabolite identification. Due to the lack of metabolite standards, metabolites were identified using exact mass and fragmentation spectra. Peak area of metabolites were extracted using MzMine 2.53 in targeted mode, and metabolites were normalized to internal standard and to the tissue weight.All statistical analyses were performed using GraphPad Prism 9.4.0 and presented as mean ± standard error of the mean (SEM). In cell experiments, nonparametric Kruskal–Wallis test followed by Dunn’s test was used to assess the significance between the treatments. Unpaired nonparametric Mann–Whitney U test was used to determine the significance between control and p-cresol-supplemented groups in the mouse experiments. Significance was established at p < 0.05.Gcg) expression at 100- (p = 0.04) and 1,000 µM concentrations (p = 0.07)  improved glucose tolerance in mice with marked increase in β-cell proliferation .TA, FB, MG conceptualized the project. PT, AV, KT performed experiments. TA, FB, TM interpreted data. PT, TA wrote the manuscript, FB, MG critically reviewed the manuscript. All authors contributed to the article and approved the submitted version.


Due to high rates of incidence and disability, postoperative cognitive dysfunction (POCD) currently receives a lot of clinical attention. Disturbance of fatty acid oxidation is a potential pathophysiological manifestation underlying POCD. Peroxisome proliferator-activated receptor α (PPARα) is a significant transcription factor of fatty acid oxidation that facilitates the transfer of fatty acids into the mitochondria for oxidation. The potential role of PPARα intervention in POCD warrants consideration.The present study is aimed to investigate whether PPARα agonist fenofibrate (FF) could protect long-term isoflurane anesthesia-induced POCD model and to explore the potential underlying function of fatty acid oxidation in the process.in vivo with C57BL/6J mice and in vitro with N2a cells. Cells and mice were pretreated with PPARα agonist FF before anesthesia, after which fatty acid oxidation and cognitive function were assessed. The level of fatty acid oxidation-related proteins was determined using western blotting. The contextual fear conditioning test was utilized to evaluate mice’s learning and memory.We established the POCD model via 6 h long-term isoflurane anesthesia In vivo results showed that preconditioned FF reached similar effects. More crucially, FF has been shown to reduce cognitive damage in mice after long-term isoflurane anesthesia. Additionally, our data showed that after blocking fatty acid oxidation by Etomoxir, FF failed to protect cognitive function from long-term isoflurane anesthesia.Our results showed that 6 h long-term isoflurane anesthesia induced contextual memory damage in mice, accompanied by decreases of fatty acid oxidation-related proteins  both in the hippocampus of POCD mice and in N2a cells. In the N2a cell model, pretreatment of PPARα agonist FF led to the upregulation of fatty acid oxidation-related proteins. Pretreatment of PPARα agonist FF can protect against long-term isoflurane anesthesia-induced POCD by enhancing fatty acid oxidation.  Most studies concerning the mechanisms of POCD have focused on inflammatory response, metabolic disturbance, oxidative stress, etc. . The metRoy et al. showed that PPARα-deficient but normal PPARβ or PPARγ hippocampal neurons had decreased calcium inflow and the expression of various synaptic plasticity-related molecules was low . PPARα, Our study focused on whether the fatty acid oxidation process was involved in the isoflurane-induced POCD and showed the effect of increased PPARα levels on fatty acid oxidation and cognitive function. We administered PPARα agonist fenofibrate (FF) to mice as pharmacological intervention. As a lipid-lowering agent, FF is a fibric acid derivative that has been widely used for the treatment of dyslipidemia in the clinic ,15. The 22.1All animal experiments were performed with the approval of the Laboratory Animal Ethics Committee of Drum Tower Hospital, and all experimental procedures complied with the ARRIVE guidelines and were conducted according to the EU Directive 2010/63/EU for animal experiments. In this study, 8-week-old male C57BL/6J mice weighing 20–22 g were used. We tried our best efforts to minimize the number of mice. All animals adapted for at least 3 weeks, with a 12 h light/dark cycle (light on at 8:00 and light off at 20:00) and appropriate room temperature (23 ± 1°C). The animals were supplied with freely available food and water.2.2Sodium carboxymethyl cellulose   was prepared with saline. FF  was dissolved in 1% CMC to obtain 75, 150, and 300 mg/kg FF solutions. Mice randomized for FF pretreatment were injected 0.01 mL FF solution per gram intragastrically 30 min before anesthesia. The mice in the Con group and the Ane group were administered with the same volume of 1% CMC. Due to its solubilization property and stability, CMC solution is used to dissolve poorly soluble medicines or deliver medicines as a pharmaceutical carrier . Also, o2.3Mice were put in Plexiglass chambers prefilled with 5% isoflurane  in 100% oxygen (2–3 L/min) for induction. They were maintained under a mixture of 1.3% isoflurane and 100% oxygen. The anesthesia operation lasted 6 h. During the whole procedure, the inspiratory concentration of isoflurane and the respiratory activity of mice were monitored automatically or manually.2.4Panlab Fear Conditioning System  was used to conduct the contextual fear conditioning test. During the training phase, mice were positioned in the chamber and were allowed to explore freely for 3 min, followed by a 30 s pulsing tone  and a 2 s mild foot shock (0.75 mA). Then, the mice remained in the chamber for 1.5 min. After 24 h, mice were positioned in the same chamber for 5 min without sound or shock for the contextual memory test. Throughout the test, Packwin 2.0 software was used to record the mice’s freezing behavior continually. The mice’s freezing behavior, an absolutely immovable posture except for breathing, was transformed to the percentage of freezing time to evaluate their learning and memory.2.52 until optimum growth, and adhesion to the surface of the plates were observed. The medium was changed daily. After 48 h, cells were then trypsin-removed from the plates, centrifuged at 800 rpm for 5 min, and lysed with NP-40 lysis buffer  with the addition of an AEBSF protease inhibitor  and stored at −80°C until further use.Mouse neuroblastoma cells (N2a cells) were plated on 24 tissue culture well plates  at 200,000 cells/well for 48 h in the tissue culture medium  at 37°C in 5% CO2.6To measure fatty acid oxidation-related protein levels , WB was performed. All mice were killed promptly following the behavioral test. The mice’s hippocampus was isolated and the tissue was stored at −80°C. In brief, the hippocampus tissue was washed and the surface water was dried. Then, it was homogenized in cold RIPA lysis buffer  with proteinase and phosphatase inhibitors  and centrifuged to obtain protein supernatants. The total amount of protein was determined by the BCA protein method . The same amount of protein (40 μg) was loaded into each lane of 10% SDS-PAGE gel, separated by electrophoresis. Subsequently, the proteins were transferred to the polyvinylidene fluoride membrane . After that, the membrane was blocked with 5% (w/v) milk for 1 h. The membranes were incubated with primary antibodies against PGC1α , CPT1A , PPARα , β-actin , and GAPDH  for 1 h, followed by HRP-conjugated secondary antibodies .2.7t-test or one-way ANOVA followed by Bonferroni post hoc tests using SPSS 17.0 software. Significance was defined when P < 0.05.All data were expressed as mean ± SD (standard deviation). Statistical analyses of both cognitive behavioral tests and WBs were conducted by Ethical approval: The research related to animals’ use has been complied with all the relevant national regulations and institutional policies for the care and use of animals. All animal experiments were performed with the approval of the Laboratory Animal Ethics Committee of Drum Tower Hospital, and all experimental procedures complied with the ARRIVE guidelines and were conducted according to the EU Directive 2010/63/EU for animal experiments.33.1aP = 0.006), Day 3 (aP = 0.002), and Day 7 (aP = 0.002) compared with the Con group. Meanwhile, to test the effects on the fatty acid oxidation pathway in POCD mice, the levels of fatty acid oxidation-related proteins PGC1α, CPT1A, and PPARα in the hippocampus of mice were measured using WB. Compared with the Con group, the protein levels of PGC1α (aP = 0.019), CPT1A (aP = 0.010), and PPARα (aP = 0.020) in the hippocampus of the Ane group were significantly reduced after anesthesia  = 0.029, aP (Ane 8 h) = 0.003, bP (Ane 8 h) = 0.043; CPT1A: aP (Ane 6 h) = 0.021, aP (Ane 8 h) = 0.005, bP (Ane 6 h) = 0.011, bP (Ane 8 h) = 0.003, cP (Ane 6 h) = 0.008, cP (Ane 8 h)= 0.002; PPARα: aP (Ane 6 h) = 0.012, aP (Ane 8 h) = 0.014). After that, 6 h isoflurane exposure was selected to establish a prolonged anesthesia cell model for the following in vitro experiments.Compared with the Con group, PGC1α, CPT1A, and PPARα protein expression levels were significantly decreased after 1.3% isoflurane intervention for 6 and 8 h rather than 2 and 4 h isoflurane exposure (aP = 0.028), CPT1A (aP = 0.001), and PPARα (aP = 0.020) in the Ane group were significantly decreased (aP = 0.006) in the FF 10 + Ane group were also significantly decreased (bP = 0.039), CPT1A (bP = 0.033), and PPARα (bP = 0.047) in the FF 20 + Ane group were significantly increased  = 0.002, bP (FF 150 + Ane) = 0.008, bP (FF 300 + Ane) = 0.019; CPT1A: bP (FF 75 + Ane) = 0.031, bP (FF 150 + Ane) = 0.033, bP (FF 300 + Ane) = 0.015; PPARα: bP (FF 75 + Ane) = 0.030, bP (FF 150 + Ane) = 0.044, bP (FF 300 + Ane) = 0.036) and Day 3 (PGC1α: bP (FF 75 + Ane) = 0.017, bP (FF 150 + Ane) = 0.009, bP (FF 300 + Ane) < 0.001; CPT1A: bP (FF 75 + Ane) = 0.046, bP (FF 150 + Ane) = 0.044, bP (FF 300 + Ane) = 0.040; PPARα: bP (FF 75 + Ane) = 0.023, bP (FF 150 + Ane) = 0.002, bP (FF 300 + Ane) = 0.002) after anesthesia , the FF 150 + Ane group , and the FF 300 + Ane group , while PGC1α protein levels only in the FF 300+Ane group were significantly higher than that in the Ane group (bP = 0.009) (Based on the results of using WB . Compareesthesia . On Day = 0.009) .3.4bP (FF 75 + Ane) < 0.001, bP (FF 150 + Ane) = 0.001, bP (FF 300 + Ane) = 0.007), Day 3 (bP (FF 75 + Ane) = 0.028, bP (FF 150 + Ane) < 0.001, bP (FF 300 + Ane) < 0.001), and Day 7 (bP (FF 75 + Ane) = 0.003, bP (FF 150 + Ane) = 0.008, bP (FF 300 + Ane) = 0.021) after anesthesia.In order to determine the effects of FF pretreatment on cognitive impairment after long-term isoflurane anesthesia, the contextual memory of mice was detected using the fear conditioning system. As shown in bP < 0.001). Remarkably, the percentage of freezing time of the ETO + FF + Ane group was significantly lower than that in the FF + Ane group (dP < 0.001), which suggests that after blocking fatty acid oxidation by ETO, FF failed to improve the memory impairment after long-term isoflurane anesthesia.To further validate whether the fatty acid oxidation pathway is involved in the neuroprotective effect of FF on anesthesia-induced memory impairment, 25 mg/kg CPT1A inhibitor ETO was intraperitoneally injected into mice for 3 days before anesthesia. Based on the results in 4in vitro and in vivo experiments showed that FF pretreatment reversed the decrease of related protein expression levels. Furthermore, our results showed that a supplement of FF could improve impairments of contextual memory after long-term isoflurane anesthesia in mice. To thoroughly explore whether FF protects against cognitive impairment after anesthesia by enhancing the fatty acid oxidation pathway, a CPT1A inhibitor, ETO, was used to block the pathway. Results showed that ETO pretreatment failed to despair the memory of POCD mice but could block the protective effect of FF. These results further suggested that FF protects memory impairment after isoflurane anesthesia by enhancing the fatty acid oxidation pathway.This study focused on the memory impairment induced by 6 h long-term isoflurane anesthesia, simulating the process of “POCD,” as shown in in vivo and in vitro [Some studies concerning the mechanisms of POCD have focused on disorders of fatty acid oxidation. Qian and Wang investigated the serum metabolism of POCD and the results revealed that fatty acid metabolism and lipid metabolism were considerably altered . Researcin vitro . Meanwhiin vitro . In POCDin vitro  when indin vitro , such asin vitro ,23. In tin vivo and in vitro in POCD models. In line with our results, Goto et al. showed that PPARα agonist FF upregulates PGC1α transcription [PPARα is altered in multiple kinds of neurodegenerative/neurodevelopmental and psychiatric disorders, suggesting that this receptor could be a viable target in novel therapeutic strategies. FF, one of the PPARα agonists, is a derivative of fibric acid and is commonly applied in the treatment of adults with dyslipidemia, primary hypertriglyceridemia, and hypercholesterolemia . Previoucription . Khorolscription . The procription . Howevercription . TherefoPPARα is reported to be critical in cognition and emotions in the study of the last decade . Roy et 2O2, and MDA with FF pretreatment following anesthesia. In this way, our study will be more convincing. Further study should be performed to solve the inadequacies listed above.There are some limitations to our research: (1) we investigated the neuroprotective effect of FF on cognitive impairments through the fatty acid oxidation pathway in the POCD model at the protein level. Considering PPARα is an important transcription factor, more research should be carried out at the transcriptional level to further verify the regulatory effects of FF on fatty acid oxidation-related proteins. (2) In each group, the sample sizes for protein detection were less than those for behavior assessment. (3) As shown in 5The present study demonstrated the protective effect of PPARα agonist FF on memory impairment after long-term isoflurane anesthesia and identified the role of fatty acid oxidation-related proteins in the process of POCD. This finding could help to improve our understanding of the amnestic effect of isoflurane and to provide new strategies for it.


FaGAPC2) or pyruvate kinase  inhibited strawberry fruit ripening and the accumulation of citric acid, leading to a range of maturity stages from partial red to full red stage. The combined transcriptome and metabolome analysis revealed that overexpression of FaGAPC2 and FaPKc2.2 significantly suppressed the expression of phosphoenolpyruvate carboxykinase  but enhanced the content of glutamine and aspartic acid. Meanwhile, the activities of PEPCK and glutamate decarboxylase (GAD) were inhibited, but the activities of glutamine synthase (GS) were increased in FaGAPC2/FaPKc2.2-overexpressed fruit. Further, functional verification demonstrated that overexpression of FaPEPCK can promote strawberry fruit ripening, resulting in a range of maturity stage from full red to dark red stage, while the citric acid synthase (CS) activities and citric acid content were significantly decreased. Overall, this study revealed that FaGAPC2/FaPKc2.2 and FaPEPCK perform an important role in reducing citric acid content in strawberry fruit, and FaGAPC2/FaPKc2.2 mainly by promoting the GS degradation pathway and FaPEPCK mainly by inhibiting the CS synthesis pathway.Citric acid is the primary organic acid that affects the taste of strawberry fruit. Glycolysis supplies key substrates for the tricarboxylic acid cycle (TCA cycle). However, little is known about the regulatory mechanisms of glycolytic genes on citric acid metabolism in strawberry fruits. In this study, the citric acid content of strawberry fruit displayed a trend of rising and decreasing from the initial red stage to the full red stage and then dark red stage. Thus, a difference in citric acid metabolic regulation was suspected during strawberry fruit development. In addition, overexpression of either cytoplasm glyceraldehyde-3-phosphate dehydrogenase (FxaC_14g13400, namely  However+ as a coenzyme specifically  and pyruvate kinase (PK) are the key enzymes of the glycolytic pathway. GAPC catalyzes the oxidative phosphorylation of glyceraldehyde-3-phosphate (G3P) to 1, 3-biphosphoglycerate , using NADifically . Besidesifically . GAPCs aifically . Recentlic acids . Besides pummelo . PEPCK i pummelo . PEPCK m pummelo . DespiteFaGAPC2/FaPKc2.2 and FaPEPCK in regulating citric acid metabolism in strawberry fruit were investigated. This study not only provides valuable genetic resources for strawberry quality improvement, but also offers new insights for further research in this field.Citrate is the predominant organic acid in strawberry fruit, and the content of total soluble solids (TSS) is not high, ranging from 7%-16%. The sour-sweet flavor is the taste sensation that most consumers experience . Moreove22.1Fragaria × ananassa cv. ‘Benihoppe’) plants were grown in a plastic greenhouse in the field (from September 2020 to March 2021) at the Teaching and Research Base of Sichuan Agricultural University in Chongzhou City, Sichuan Province, China. Fruit developmental stages were defined as small green (SG), large green (LG), white (W), initial red (IR), partial red (PR), full red (FR) and dark red (DR) stage based on days post-anthesis (DPA) and the color of receptacles (Agrobacterium injection. Fruit samples (including seeds) were taken only from the middle part of the fruit, and the medullary part was removed . The first-strand cDNA was synthesized using the Revert Aid H Minus Reverse Transcriptase  with random primers. All PCR amplifications were conducted on a PT100 thermal cycler . Reactions of 20 μL were set up by a combination of 1 μL of cDNA template, 1 μL of forward and reverse gene-specific primer, and 10 μL of PrimeSTAR Max Premix . A total of 34 PCR cycles of reactions were employed, consisting of one step of 98°C 3 min, 98°C 10s, 60°C 15 s, 72°C 1 min, and a last extension step of 72°C for 5 min. PCR amplicons were detected through electrophoresis in a 1% agarose gel.2.3FaGAPC2/FaPKc2.2 and FaPEPCK were amplified with the primer pair OE-FaGAPC2-F/OE-FaPKc2.2-F, OE-FaPEPCK-F and OE-FaGAPC2-R/OE-FaPKc2.2-R, OE-FaPEPCK-R  and a pocket refractometer , respectively. The titratable acid (TA) content was determined by an acid-base titration method with 0.1 M NaOH . The results were expressed as citric acid % (w/v). The sucrose, fructose, glucose, citric acid and malic acid contents were detected using the HPLC method (2-RP column using Agilent HPLC system (eluent program: 75% acetonitrile 10 min) subsequently. Organic acid determination: 0.5 g strawberry fruit was extracted with 4 mL 0.2% phosphoric acid water, and detected by an Athena C18-WP column using Agilent HPLC system (eluent program: 3% methanol and 97% phosphoric acid water (0.2%) phosphoric acid water,10 min) subsequently. The content of sugar and organic acid was quantified by comparing them with the corresponding external standards. All HPLC-grade standards were purchased from Sigma (USA). Experiments were independently repeated three times.Fruit color was measured using a chroma meter . The results were presented as C method . Sugar d2.5The pH differential method  was used2.6The GS, GAD, PEPCK, CS, ACL, IDH enzyme activity, ABA and IAA content was measured by enzyme-linked immunoassay (ELISA) kit . Strawberry fruit (1.0 g) was extracted with 10 mL 80% ethanol (v/v) at 4°C for 1 h and 10 mL phosphate buffer (PBS), respectively. The PBS-extracted supernatant and 80% ethanol-extracted supernatant were used for the determination of enzyme activity and ABA/IAA content, respectively. Add 50 μL of supernatant diluted 5 times and 100 μL of enzyme standard reagent to the enzyme standard plate, incubate for 1 h at 37 °C. Repeat washing the solution for 5 times, pat dry and add color developer A (50 μL) and B (50 μL), mix well, and then develop color at 37 °C for 15 min. Add 50 μL of termination solution and measure absorbance at 450 nm within 15 min. Add 50 μL of each standard at different concentrations to the standard wells, and add no sample and enzyme standard reagent to the blank control wells. The rest of the operation is the same. The experiment was repeated three times.2.7p < 0.05 were included in the statistical analyses. Six independent replications were included for each sample.The fruit samples were ground and extracted with 1.0 mL 70% aqueous methanol. After centrifugation at 10,000 × g for 10 min, the extract was filtered and quantified at Novogene . UHPLC-MS/MS analyses were performed using a Vanquish UHPLC system coupled with an Orbitrap Q ExactiveTMHF-X mass spectrometer . Samples were injected onto a Hypesil Gold column  using a 17 min linear gradient at a flow rate of 0.2 mL/min. Eluent A (0.1% FA in Water) and eluent B (methanol) were used in the positive polarity mode. The eluents for the negative polarity mode were eluent A  and eluent B (methanol). The solvent gradient was set as follows: 2% B, 1.5 min; 2-100% B, 12.0 min; 100% B, 14.0 min; 100-2% B, 14.1 min; 2% B, 17 min. Q ExactiveTMHF-X mass spectrometer was operated in positive/negative polarity mode with a spray voltage of 3.2 kV, the capillary temperature of 320°C, sheath gas flow rate of 40 arb and aux gas flow rate of 10 arb. The raw data was processed using the Compound Discoverer 3.1 (CD3.1) system. Differential metabolites with a percentage of coefficient of variation (%CV) below 30%, variable importance in the projection (VIP) >1.0, fold changes (FC) >1.5 and the adjusted 2.8FaGAPC2/FaPKc2.2 was efficiently overexpressed was used for RNA-seq library construction. The ones from fruits infiltrated with the empty vector were used as control. Library constructions were done following the standard protocol of Illumina Next®UltraTM RNA Library Prep Kit . A total of six (three replication for each treatment) libraries were clustered and sequenced  by Novogene  on a Hiseq-2500 platform.Total RNA isolated from three groups of fruits in which 2 transformed fold change (log2FC) greater than 0.5 or less than -0.5) and the adjusted p-value ≤ 0.05 were considered as differentially expressed. KEGG pathway and GO enrichment analysis were conducted using the R package cluster Profiler (v4.2.0)  bases and adaptors by utilization trim-galore (v0.6.6). The cleaned reads were mapped onto the strawberry genome and quantified using the accurate Fanse3 mapping pipeline  with par(v4.2.0) .2.92O and 10 μL of TB Green Premix Ex Taq II . 40 circles of three-step PCR reactions were carried out with a denaturation step at 94°C for 3 min, 94°C for 30 s, an annealing step at 58°C for 10 s and an extension at 72°C for 10 s. The FaActin2 gene (LOC101313255) served as the internal control. The relative mRNA expression level of the target genes was calculated by using the 2−△△Ct method. Three biological and three technological replications were done for all the RT-qPCR reactions.A real-time quantitative PCR assay was performed to measure the expression level of the selected genes in strawberry fruit samples. A 20 μL reaction system was established, including 1 μL of cDNA template, 1 μL of gene-specific primer pairs .Data analyses were conducted with IBM SPSS Statistics software (Version 25.0). Results were expressed as mean ± SD. 33.1The ripening process of strawberry fruit was accompanied by continuous accumulation of anthocyanin and dynamic changes of organic acid content. To elucidate their characteristics in strawberry fruit development, the content of citric acid and pelargonidin-3-glucoside was determined. The results showed that pelargonidin-3-glucoside started to accumulate from the initial red stage, reaching a maximal level of 244.96 μg/g at the dark red stage, 1.63-fold higher than that at the full red stage  was employed for metabolite analysis. To test the differences in metabolic between profiles groups and sample replicates within the group, we carried out principal component analysis (PCA) including 18 samples . Meanwhile, FaGAPC2 vs. CK and FaPKc2.2 vs. CK shared 112 common differential metabolites  were detected in the FaGAPC2 and FaPKc2.2 overexpressed fruit, respectively  and α-ketoglutarate (1.55- and 1.05-fold), but increased the relative levels of glutamine (0.3- and 0.58-fold) and asparagines (0.84- and 0.74-fold), respectively (FaPEPCK (FxaC_1g21491) involved in mediating the decarboxylation of oxaloacetate (OAA) to generate phosphoenolpyruvate (PEP) was detected as common DEG in Glycolysis-Krebs cycle pathway, which was downregulated 2.2- and 3.2-fold in FaGAPC2/FaPKc2.2-overexpressed fruit compared to that of the control, respectively , gluconeogenesis (FaPEPCK), citrate synthase (FaCS) and citrate degradation   Figure 8https://db.cngb.org/cnsa/home/) with accession number CNP0002459 and CNP0004133.All RNAseq reads were deposited in the CNGB nucleotide sequence archive (YL and HT: designed the research. MY, GH, and YP: performed the experiments. LW, XL, and YJ: data collation. CH, MS, and MZ: data analysis. QC, ML, YZ, YL, and ZY: provided analysis support. YW, WH, and XW: data visualization. MY and YL: drafted and revised the manuscript. YL and HT conceived the project. All authors contributed to the article and approved the submitted version.


T, an anaerobic intestinal bacterium that potentially accumulates acetaldehyde at levels exceeding its minimum mutagenic concentration (50 µM) in the colon and rectum, was isolated from the feces of a patient with alcoholism. The 16S rRNA gene sequence of strain C5-48T showed high similarity to the corresponding sequences of Lachnoclostridium edouardi Marseille-P3397T (95.7%) and Clostridium fessum SNUG30386T (94.7%). However, phylogenetic analysis using the sequences of the 16S rRNA, rpoB, and hsp60 genes and whole-genome analysis strongly suggested that C5-48T should be included in the genus Enterocloster. The novelty of strain C5-48T was further confirmed by comprehensive average nucleotide identity (ANI) calculations based on its whole-genome sequence, which showed appreciable ANI values with known Enterocloster species . The temperature range for growth of strain C5-48T was 15–37 °C with an optimum of 37 °C. The pH range for growth was 5.5–10.5 with an optimum of 7.5. The major constituents of the cell membrane lipids of strain C5-48T were 16:0, 14:0, and 18:1 ω7c dimethyl acetal fatty acids. On the basis of the genotypic and phenotypic properties, Enterocloster alcoholdehydrogenati sp. nov. is proposed, with the type strain C5-48T (= JCM 33305T = DSM 109474T).Strain C5-48The online version contains supplementary material available at 10.1007/s00284-023-03285-1. Chronic consumption of excess alcohol induces colonic lesions, colonic inflammation, and oxidative stress in the gut, increasing the risk of colorectal cancer , 2. Acet. comprehensively examined the ability of bacterial isolates from the feces of patients with alcoholism to produce and decompose AcH . ForT is shown in Table L. sphenoides ATCC 19403T and E. clostridioformis JCM 1291T. The major cellular fatty acids of strain C5-48T were 16:0, 14:0, and 18:1 ω7c dimethyl acetal (DMA), and these levels were much higher than those of the other two strains. C5-48T showed lower 18:1 ω9c and 18:1 ω9c DMA contents, which were the major fatty acids in the other two strains  is a rod-shaped, non-endospore-forming, anaerobic organism. The cells stain Gram-positive. The rods measure 3–4 × 1 µm. The cell wall mainly comprises 16:0, 14:0, and 18:1 ω7c DMA fatty acid. The temperature range for growth is 15–37 °C with an optimum of 37 °C. The pH range for growth is 5.5–10.5 with an optimum of 7.5. The strain is slightly salt-tolerant and can grow in the presence of 10.5% (w/v) NaCl in medium A.2S production, and the Voges–Proskauer reaction, but negative for indole production, lysine decarboxylase, ornithine decarboxylase, urease, and gelatinase activities. Acid can be produced from L-arabinose, L-rhamnose, glucose, sucrose, melibiose, D-mannitol, D-sorbitol, inositol, and amygdalin. The G + C content of the DNA is 49.4 mol%  or 48.8% (based on the whole-genome sequence). The type strain is C5-48T, which was isolated from the feces of a patient with alcoholism. The type strain has been deposited in the following culture collections: Riken BioResource Research Center (as JCM 33305T) and Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (as DSM 109474T). Strain C5-48T represents a novel species of the genus Enterocloster, and the name Enterocloster alcoholdehydrogenati sp. nov. is proposed.The bacterium is positive for β-galactosidase activity, arginine dihydrolase activity, tryptophan deaminase activity, HSupplementary file1 (PDF 1427 KB)Supplementary file2 (TXT 74 KB)Supplementary file3 (TXT 143 KB)Supplementary file4 (TXT 62 KB)Below is the link to the electronic supplementary material.


Chinese Nong-favor daqu, the presentative liquor starter of Baijiu, has been enriched with huge amounts of enzymes in degrading various biological macromolecules by openly man-made process for thousand years. According to previous metatranscriptomics analysis, plenty of α-glucosidases were identified to be active in NF daqu and played the key role in degrading starch under solid-state fermentation. However, none of α-glucosidases was characterized from NF daqu, and their actual functions in NF daqu were still unknown.Escherichia coli BL21 (DE3). NFAg31A exhibited the highest sequence identities of 65.8% with α-glucosidase II from Chaetomium thermophilum, indicating its origin of fungal species, and it showed some similar features with homologous α-glucosidase IIs, i.e., optimal activity at pH ~ 7.0 and litter higher temperature of 45 ℃, well stability at 41.3 ℃ and a broad pH range of pH 6.0 to pH 10.0, and preference on hydrolyzing Glc-α1,3-Glc. Besides this preference, NFAg31A showed comparable activities on Glc-α1,2-Glc and Glc-α1,4-Glc, and low activity on Glc-α1,6-Glc, indicating its broad specificities on α-glycosidic substrates. Additionally, its activity was not stimulated by any of those detected metal ions and chemicals, and could be largely inhibited by glucose under solid-state fermentation. Most importantly, it exhibited competent and synergistic effects with two characterized α-amylases of NF daqu on hydrolyzing starch, i.e., all of them could efficiently degrade starch and malto-saccharides, two α-amylases showed advantage in degrading starch and long-chain malto-saccharides, and NFAg31A played the competent role with α-amylases in degrading short-chain malto-saccharides and the irreplaceable contribution in hydrolyzing maltose into glucose, thus alleviating the product inhibitions of α-amylases.An α-glucosidase , the second highest expressed α-glucosidases in starch degradation of NF daqu, was directly obtained by heterologous expression in This study provides not only a suitable α-glucosidase in strengthening the quality of daqu, but also an efficient way to reveal roles of the complicated enzyme system in traditional solid-state fermentation. This study would further stimulate more enzyme mining from NF daqu, and promote their actual applications in solid-state fermentation of NF liquor brewing, as well as in other solid-state fermentation of starchy industry in the future.The online version contains supplementary material available at 10.1186/s12934-023-02124-z. Baijiu (Chinese liquor), one of the oldest distilled spirits in the world, has been produced for thousands of years, and Nong-favor  baijiu accounts for about 70% of baijiu production . NF daquStarch is the highest ingredients of cereal material in NF daqu, and its degradation requires synergistic actions among various enzymes, such as α-amylase, β-amylase, glucoamylase, α-glucosidase, isoamylase and pullulanase , making p-nitrophenyl-α-D-glucopyranoside (pNPG)), whereas Type II and III AGs prefer the hydrolysis of homogeneous linkages . Between Type II and III AGs, Type III AGs show higher efficiency on degrading polysaccharides, such as starch. According to the CAZy classification, AG (EC 3.2.1.20) are reported in GH4, GH13, GH31, GH63, GH97, and GH122, and mainly belong to GH13 and GH31. AG in GH31 family prefers the degradation of homogeneous substrates (type II and Type III), whereas AG in GH13 family prefers the degradation of heterogeneous substrates (type I) [α-Glucosidase (AG) EC 3.2.1.20) could hydrolyze α-1,4-glucosidic linkages to release glucose from non-reducing end via an exo-acting manner –15. AGs (type I) . Most im(type I) . Thus, t(type I) , 18, α-1(type I) , α-galac could hy(type I) , sucrase(type I) , α-N-ace(type I) , 25.NF daqu is a typic medium-temperature daqu, which must be maintained at a relatively high temperature of 50 − 60 °C for more than one week during its making process , 9. AccoEscherichia coli Trans5ɑ and BL21(DE3)  was used for plasmid maintenance and protein expression, respectively. T4 DNA ligase, NdeI, HindIII  and pET-28a + vector  were used for gene cloning. Ni-NTA Sefinose™ Resin was purchased from Sangon Biotech  for protein purification. p-Nitrophenyl-α-D-glucopyranoside (pNPαG), p-Nitrophenyl-β-D-glucopyranoside (pNPβG), p-Nitrophenyl-β-D-xylopyranoside (pNPβX), p-Nitrophenyl-α-D-mannopyranoside (pNPαM), and p-Nitrophenyl-α-L-arobinopyranoside (pNPαA) were from J&K Scientific , while soluble starch, malto-oligosaccharides, Nigerose, Kojibiose, Isomaltose, Sucrose, Kanamycin sulphate, imidazole, isopropylthio-β-galactoside (IPTG) and other reagents were purchased from yuanye Bio-Technology .NdeI and HindIII site, respectively. The PCR products of NFAg31A and pET-28a + vector were both cleaved by NdeI and HindIII, and their purified DNA fragments were ligated with T4 DNA ligase (New England Biolabs). The recombinant plasmid of pET-28a+-NFAg31A was then transformed into E.coli Trans5ɑ, and transformants were sprayed onto Luria-Bertani (LB) medium with kanamycin (50 µg/mL). Single colonies were re-cultured into LB medium with same antibiotics overnight, and plasmid was extracted from culture with Qiagen mini-prep kit accordingly. Finally, the recombinant plasmid with the correct inserts was verified by DNA sequencing (Sangon Biotech).Since the gene product of ORF15963 was the first characterized alpha-glucosidase in the GH31 family from NF daqu, it was designated as NFAg31A. The gene fragment of NFAg31A, excluding the signal peptide, was directly amplified from cDNA library of NF daqu by Mix (Green) , with two specific primers of NFAg31AF 5’CATATGGTGAAGCATGAAAACTTCAAGAAATG 3’ and NFAg31AR 5’AAGCTTCTAGATCCTCCACTCCCTG3’ containing E.coli BL21(DE3). In short, 1 mL pre-culture of NFAg31A transformants was inoculated into 1 L of LB medium with same antibiotics, and cultured at 37 ℃. When the culture reached an optical density of 0.6 at 600 nm, the expression was induced with 0.05 mM IPTG at 25 ℃ for 48 h. The cells were collected by centrifugation at 4 ℃, suspended in binding buffer , and disrupted by sonication with Sonics Vibra-Cell™ . The ruptured cells were clarified by centrifugation at 4 ℃, and the resulting supernatant was then applied to protein purification.Next, the recombinant protein of NFAg31A was produced in NFAg31A with C-terminal 6xHis-tag was adsorbed on Ni-NTA Sefinose™ resin, washed by binding buffer containing 20 mM imidazole, and eluted by binding buffer containing 200 mM imidazole. The elution was successively collected in fraction, and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Fractions with the purified proteins were finally concentrated to a protein storage buffer  by Amicon® Ultra-30 Centrifugal Filter Devices . Concentrations of the purified protein were determined by NanoDrop 2000c , using its extinction coefficient of 204,675 1/(mol*cm).pNPαG, pNPβG, pNPαM, pNPβX, or pNPαA in phosphate buffer (pH 6.5) at 40 ℃ for 1 h to identify its possible activities. Then, the influence of pH on activity of NFAg31A was detected by incubating 0.25 µM enzyme with 2 mM pNPαG at 45 ℃ for 30 min between 5.5 and 9.5 , while the influence of temperature on its activity was measured at its optimal pH for 30 min, under different temperatures ranging from 31 ℃ to 85 ℃. Unless otherwise specified, the standard activity of NFAg31A was performed by incubating 0.25 µM enzyme with 2 mM pNPαG at pH 7.0 and 45 ℃ for 30 min and terminating the reaction by heating at 100 ℃ for 10 min throughout this work, and the p-nitrophenol liberated was measured at 410 nm.First, 1 µM purified NFAg31A was incubated with 2 mM pNPαG at pH 7.0 and 45 ℃ for 30 min as described above. Meanwhile, the effect of temperature on stability of NFAg31A was measured by incubating 7.5 µM enzyme at different temperatures (from 35 ℃ to 51 ℃) for 30 min, and its residual activities were then measured in a same way. In addition, thermostability of NFAg31A at 39 ℃, 45 ℃ and 50 ℃ were detected by incubating 7.5 µM enzyme at those temperatures for different periods, and standard assay was performed to detect its residual activities.The effect of pH on stability of NFAg31A was determined by incubating 7.5 µM enzyme without substrates at room temperature for 30 min under different pH ranging from 3.0 to 12.0, and its residual activities were subsequently determined with 2 mM pNPαG as substrates. The activity determined in the absence of additives was taken to be 100%, and those relative activities with additives were calculated as a percentage of its activity.The effects of various metal salts and chemicals on activity of NFAg31A were detected by the incubation of 0.25 µM purified enzyme with those additives at 1 mM or 10 mM concentration under standard assay, using 2 mM pNPαG, 2 mM oligosaccharides , and 2 mg/ml soluble starch, or incubating 4 µM enzymes with 2 mM isomaltose and sucrose, at pH 7.0 and 45 ℃ for 15 min, respectively. The reaction was terminated by heating at 100 ℃ for 10 min. Same as previous study [p-nitrophenol liberated from pNPαG was measured at 410 nm with a molar extinction coefficient of 55,560 M− 1 cm− 1. One unit of the enzyme activity was defined as the amount of enzyme required to release 1µmol glucose equivalent from substrates per minute under its standard conditions. All reactions were performed in triplicate.The specific activities of NFAmy13A were detected by incubating 0.25 µM enzymes with 2 mM us study , the glupNPαG, 0.5–40 mg/ml soluble starch, 2–24 mM isomaltose, respectively. The velocities were kept in a constant scope within this time frame, and low concentration of 0.25 µM enzyme was used on pNPαG, malto-oligosaccharides, nigerose, kojibiose and soluble starch, except for 4 µM enzyme on isomaltose. The kinetic parameters were estimated by fitting data to the Michaelis-Menten equation using a nonlinear regression method (Origin 9). To estimate kinetic parameter on soluble starch, the molar concentration was converted by assuming that the average degree of polymerization per one non-reducing end of a starch chain is 43.5 [The kinetic experiments were performed by incubating the purified NFAmy13A at 45 ℃ and pH 7.0 for 15 min with increasing concentrations of substrates, i.e., 0.5–40 mM malto-oligosaccharides , 0.25–24 mM nigerose and kojibiose, 0.1–32 mM  is 43.5 .pNPαG at 45 ℃ and pH 7.0 for 30 min, containing the increasing concentrations (0.5–50 mM) of monosaccharides, i.e., glucose, mannose, xylose, fructose, galactose, arabinose. The relative activity was calculated as a percentage of the activity without the addition of monosaccharide under same condition.The possible inhibition of monosaccharides on activity of NFAmy13A was determined by incubation of 1 µM purified enzyme with 2 mM Time-course degradation of malto-oligosaccharides was measured by incubating 2 µM NFEg31A with high concentration of 5 mM malto-oligosaccharides at 45 ℃ and pH 7.0 for various times . Then, the glucose and malto-oligosaccharides released were detected by HPAEC-PAD under same condition as described above.The possible synergistic effect among NFEg31A and two previously characterized α-amylases (NFAmy13A and NFAmy13B) was studied here, since all of them were obtained from Nong-flavor daqu. Different molar concentrations and ratios were selected among them, i.e., 1:1:1 (0.5 µM NFAmy13B: 0.5 µM NFAmy13A : 0.5 µM NFEg31A), 3:7:10 (0.3 µM NFAmy13B : 0.7 µM NFAmy13A : 1 µM NFEg31A), and 1:19:20 (0.05 µM NFAmy13B : 0.95 µM NFAmy13A : 1 µM NFEg31A), so as to deeply compare their synergistic degradation of starch in daqu system. Then, different combinations of three enzymes, i.e., single enzyme , two enzyme combinations , three enzyme combination , were applied to the degradation of 1 mg/ml wheat starch at 45 ℃ and pH 7.0 for 30 min, respectively.NCBI-BLAST database searches were used to identify homologous α-glucosidases, and those characterized enzymes were finally selected for construction of phylogenetic tree. Accession numbers for all those selected enzymes were listed in Supplemental Table The nucleotide sequence of NFEg31A was deposited in the NCBI GenBank database with Accession Number of OP756524.http://pfam.sanger.ac.uk/). According to analysis on SignalP 6.0 Server , a signal peptide of 30 aa was predicted in its gene product, and without signal peptide, NFAg31A had a predictive molecular mass of 105.6 kDa.The ORF15963 showed high expression level with an RPKM value of 127.1, and its gene product (NFAg31A) was the 25th highest expression carbohydrate-active enzymes at N3 stage of NF daqu  as intracellular soluble form. Without C-terminal DUF5100 domain, NFAg31A expressed as intracellular insoluble form (data not shown). Then, the recombinant NFAg31A was purified by affinity chromatography using Ni2+-NTA resin. The purified NFAg31A showed a clear band at above 100 kDa, in good agreement with its predictive value (105.6 kDa)  Fig. b.pNPαG, not on pNPβG, pNPβX, pNPαM, or pNPαA (data not showed), indicating the α-glucosidase activity of NFAg31A. As shown in Fig. 2 + and Mg2+, while a clear inhibition was observed for most of those detected additives, such as slight inhibition of Ca2+, Ni2+, Al3+, K+ and Li+, moderate inhibition of Al3+, Mn2+, EDTA·Na2 and NH4+, severe inhibition of Zn2+, Hg2+ and SDS, and complete inhibition of Fe3+ and Cu2+  were not determined and only kcat/Km values were determined at sufficiently low substrate concentration (Table (kcat/Km) of 14.2 1/(s*mM), and isomaltose displayed the lowest kcat/Km value of 0.0014 1/(s*mM). Furthermore, among di-saccharides, kojibiose showed the lowest Km value of 3.9 mM, which could indicate a strong binding affinity between NFAg31A and kojibiose. Meanwhile, NFAg31A displayed a dramatically higher kcat value of 29.3 1/s on nigerose, which was 6.7 and 9.2 folds of that on maltose and kojibiose, respectively, which would be the main reason that NFAg31A exhibited higher catalytic efficiency on nigerose than on kojibiose or maltose. In addition, pNPαG, the unnatural substrate, displayed a relatively low kinetic values and specific activity, when compared to oligosaccharides and starch, except isomaltose  were released from starch by NFAmy13B than 0.61 mM by NFAmy13A Table , and theStrangely, NFAmy13B released higher amount (1.40 mM) of reducing products than that 0.57 mM) of NFAmy13A . A similar signal peptide with same size (30 aa) and high similarities (> 70%) was also found in putative AGIIs from fungal species, such as Aspergillus clavatus (XP_001276182.1), Penicillium manginii (KAJ5741752.1), Penicillium cataractarum (KAJ5381475.1) and Aspergillus avenaceus (KAE8150115.1). And without the same size peptide, C.thermophilum AGII was also successfully obtained by heterologous expression in E. coli BL21-CodonPlus (DE3) and folded correctly [Paecilomyces variotii, and the highest similarity (65.8%) with characterized AGII from C.thermophilum [C.thermophilum AGII and Saccharomyces cerevisiae AGII, NFAg31A was clustered to a group of α-glucosidases II (AGIIs), and NFAg31A showed a similar molecular size (105.6 kDa) with its homologous AGIIs, within size ranges from 98 kDa to 125 kDa [The gene products of ORF 15,963 was predicted to encode a signal peptide of 30aa in front of the mature protein NFAg31A), and without this signal peptide, NFAg31A was successfully obtained with active form by heterologous expression in orrectly . Thus, tum KAJ53875.1 and um KAJ53875.1 and XP_0012762.1, Peni 125 kDa –31.A, and wiApis cerana AGII [Acremonium implicatum AG [Lactobacillus johnsonii AG [Schizosaccharomyces pombe AGII [A.cerana AGII [A.cerana AGII and archaebacterial AGs. Moreover, NFAg31A was a neutral α-glucosidase, exhibiting well stability over a broad pH range of pH 6.0 to pH 10.0, which was similarly detected as pH 6.2 to pH 9.1 for S.pombe AGII and pH 5.0 to pH 8.0 for L.johnsonii AG. Therefore, NFAg31A showed similar enzymatic characterization over temperature and pH as its close members in GH31-1 subfamily, e.g., A.implicatum AG and S.pombe AGII. In short, based on both of its sequnce alignment and enzymatic characterization analysis, NFAg31A should be a typic fungal member in GH31-1 subfamily.NFAg31A showed optimal activity at 45 ℃, which was similarly detected as 45 ℃ of ana AGII  and 50 ℃catum AG , litter catum AG , 34–37, catum AG , 38–40. sonii AG  and 40 ℃ana AGII . Whereasana AGII , 30, 41,ana AGII , 42, andana AGII , 43, 44,S.cerevisiae AGII in GH31-1 subfamily [Sulfolobus acidocaldarius AG [Thermoplasma acidophilum AG [2+. Furthermore, its activity was strongly inhibited by SDS, Cu2+, and Zn2+, which was same as that of S.acidocaldarius AG and T.acidophilum AG.Meanwhile, same as its close member of ubfamily , the actarius AG  and Therhilum AG  were botpNPαG, indicating that it should be a type III AG [A.cerana, Sporothrix schenckii and S.pombe, human MGAM and SI, and AGs from L.johnsonii, Lactococcus lactis, S.solfataricus and Pyrobaculum arsenaticum. Moreover, same as its homologous α-glucosidases II (AGIIs) from C.thermophilum [S.cerevisiae [Porphyridium sp. (Rhodophyta) [Bombyx mori and Spodoptera frugiperda [L.lactis, A.implicatum and L.johnsonii  , Bombyx ugiperda , NFAg31Aii Table . Besidessite + 1 , 14. For AG Fig. . LlAG ke AG Fig. , which clc Table . Thus, Neraction , 14, whiA.implicatum AG, completely different with S.acidocaldarius AG, A.niger AG, L.johnsonii AG and S.solfataricus AG. The similar preference of human CtMGAM for longer oligosaccharides was speculated by an extra 21 amino acids in human CtMGAM and CtSI [8 domain of CtMGAM was likely responsible for this preference [Most importantly, NFAg31A showed definitely higher degrading capacity on long-chain starch than on short-chain oligosaccharides, which was same observed for Sugar beet AG and and CtSI , which meference , which wPorphyridium purpureum [Candida albicans [The addition of monosaccharides definitely reduced the activity of NFAg31A, especially glucose. Indeed, glucose was also reported to largely inhibit the activity of AGII from urpureum  and Candalbicans . Whereasalbicans , 47, whialbicans . Meanwhialbicans , thus itThe hydrolyzing pattern of NFAg31A was identified by time-course degradation of malto-oligosaccharides. Within short incubation of 1 h, NFAg31A definitely showed preference on removing glucose from all malto-oligosaccharides (M2 to M5). This finding again proved that NFAg31A is a typical GH31-type α-glucosidase, which could hydrolyze malto-oligosaccharides in an exo-acting manner, efficiently trimming glucose moiety from the non-reducing end of malto-oligosaccharides –15. As mPreviously, NFAmy13A and NFAmy13B, from same stage of Nong-flavor daqu, have showed synergistic effect in hydrolyzing starch. Here, a dramatically synergistic effect was further observed among NFEg31A, NFAmy13A and NFAmy13B in starch degradation, according to both reducing products and end products, which might result from their different hydrolyzing patterns . In detaImportantly, α-glucosidase is the most abundant enzymes involved starch degradation in NF daqu and Jiang-flavor daqu . MoreoveIn present study, a fungal α-glucosidase in GH31-1 subfamily, with relatively high expression in NF daqu, was successfully obtained and identified with high catalytic efficiencies towards α-1,3-, α-1,4-, and α-1,2-glycosidic linkages. Its synergistic and irreplaceable contribution to the degradation of starch in NF daqu was also confirmed. NF daqu is a complicated enzyme system, which is enriched with huge amounts of enzymes in hydrolyzing various α-glycosidic substrates. Therefore, the broad substrate specificity and well synergistic function of NFAg31A provide chance for enhancing the degrading capacity of NF daqu, and improving the quality and production of NF baijiu.Below is the link to the electronic supplementary material.Table S1. Accession numbers of all those selected enzymes for Phylogenetic tree analysis in four subgroups of GH31 family. Table S2. The top 30 highest expression of genes related to carbohydrate-active enzymes at the highest temperature stage (N3) of NF daqu. Table S3. The relative high expression of alpha-glucosidase (GH31) genes at the high temperature stage (N3), and their relative expressions at the beginning stage (N1), the increasing temperature stage (N2) and mature stage (N4) of NF daqu. Table S4. Effect of metal ions and chemicals on the activity of NFAg31A. Table S5. The synergistic hydrolysis of soluble starch by different molar ratios and combinations of NFAg31A and two alpha-amylases (NFAmy13A and NFAmy13B). Table S6. The end products of synergistically hydrolyzing soluble starch by NFAg31A and two alpha-amylases (NFAmy13A and NFAmy13B) at molar ratio of 3:7:10. Figure S1. Schematic representation and purification of NFAg31A. Figure S2. Sequence alignment among NFAg31A and its homologous ?-glucosidases. Figure S3. Thermostability of NFEg31A at 39 ℃, 45 ℃ and 50 ℃Supplementary Material 1:


Despite long-term integrated control programs for Eimeria stiedai infection in China, hepatic coccidiosis in rabbits persists. Th1, Th2, Th17, Treg, Th9, and Th21 cells are involved in immune responses during pathogen infection. It is unclear whether Th cell subsets are also involved in E. stiedai infection. Their roles in the immunopathology of this infection remain unknown. Therefore, monitoring these T-cell subsets’ immune responses during primary infection of E. stiedai at both transcriptional (mRNA) and protein (cytokines) levels is essential.In experimentally infected New Zealand white rabbits, mRNA expression levels of their transcript—TBX2 (Th1), GATA3 (Th2), RORC (Th17), Foxp3 (Treg), SPI1 (Th9), and BCL6 (Th21)—were evaluated using quantitative real-time polymerase chain reaction (qRT-PCR), whereas Th1 (IFN-g and TNF-a), Th2 (IL4), Th17 (IL17A and IL6), Treg (IL10 and TGF-b1), Th9 (IL9), and Th21 (IL21) cytokines were measured using enzyme-linked immunosorbent assays (ELISAs).We found that levels of TBX2, GATA3, RORC, SPI1, and BCL6 in the livers of infected rabbits were elevated on days 5 and 15 post-infection (PI). The concentrations of their distinctive cytokines IFN-g and TNF-a for Th1, IL4 for Th2, IL17A for Th17, IL9 for Th9, IL21 for Th21, and IL10 for Treg IL10 were also significantly increased on days 5 and 15 PI, respectively (p < 0.05). On day 23 PI, GATA3 with its cytokine IL4, RORC with IL17A, Foxp3 with IL10 and TGF-b1, and SPI1 with IL9 were significantly decreased, but TBX2 with IFN-g and IL6 remained elevated.Our findings are the first evidence of Th1/Th2/Treg/Th17/Th9/Th21 changes in E. stiedai-infected rabbits and provide insights into immune regulation mechanisms and possible vaccine development. Eimeria stiedai. First discovered more than 100 years ago, it has a high host specificity, infectivity, and lethality. Post-weaning rabbits are particularly susceptible, with mortality as high as 80% , it was found that the transcription factor TBX2 was positively correlated to TNF-α, but not to IFN-γ, which was correlated to another Th1 transcriptional factor, IRF8 , and cytokines  in rabbits infected with E. stiedai.T helper cells with specific cytokines  play essential roles in parasitic diseases . Usuallyor, IRF8 ; thus, oSchistosoma mansoni-infected mice, higher expression levels of hepatic IL17A were found in CBA mice, suggesting that its lesions may be associated with IL17A produced by Th17 cells  and orchestrates the Th1 cell migration by regulating the expression of chemokines and chemokine receptors. TBX2 and GATA3 regulate Th1/Th2 cell fate using a synchronous mechanism, by directly binding to the genetic locus of cytokines: TBX2 binds IFN-γ whereas GATA3 binds IL4 and other Th2 cytokines such as IL5 and IL13, leading to the expression of these proteins  development in the thymus or in the presence of TGF-β1 and retinoic acid. High levels of IL17 is expressed by Th17 under the transcriptional regulation of RORC. In some studies, it was suggested that the signature transcription factor RORC of Th17 cells is also induced by TGF-β1, thus leading to differentiation of Treg and Th17 lineages. In the absence of a second signal derived from proinflammatory cytokines, Foxp3 can inhibit RORC function and promote Treg differentiation. However, when cells also receive signals from proinflammatory cytokines , the function of Foxp3 is inhibited and Th17 differentiation is induced. Thus, it is the balance between Foxp3 and RORC function that determines the fate of CD4+T cells and the type of immune response that would be generated  is a transcription factor that belongs to the E-twenty six (ETS) family of proteins and is a key factor for the differentiation of the IL9-producing Th9 subset, in humans and mice   . While B B cells .E. stiedai infection, it is unclear whether different mechanisms are involved. For these reasons, we measured dynamic changes in Th cells, including Th1, Th2, Th17, Treg, Th9, and Th21, during different stages of E. stiedai infection. Our findings provide a theoretical basis to control for protective and therapeutic vaccinations.In This study was carried out in full compliance with the framework of noncommercial scientific research, authorized by the Animal Ethics Committee of Sichuan Animal Science Academy .E. stiedai strain in this study was provided by the Department of Parasitology at Sichuan Agricultural University. Three coccidia-free New Zealand white rabbits (5 weeks old) were orally infected with E. stiedai to obtain more oocysts. Then high numbers of unsporulated oocysts were harvested from liver with grinding, filtration, and centrifugation. Finally, the oocysts were sporulated in 2.5% potassium dichromate solution at 28°C and stored at 4°C.The n = 18, six rabbits for each time point) and an experiment group . They received 1.0 × 105E. stiedai orally and were euthanized on days 5, 15, and 23 post-infection (PI). All rabbits were monitored for 5 days by testing the daily collected fecal samples to confirm the infection. Blood samples were collected for quantitative real-time polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assays (ELISAs).We divided 36 female New Zealand white rabbits aged 50 days into a control group  as follows: 4 μl of RNA, 2 μl of 5× gDNA digester Buffer, 1 μl of gDNA, 10 μl of 2× Hifar® II SuperMix plus, and 3 μl of sterile RNase-free water. The amplification conditions were 25°C for 5 min, 42°C for 30 min, and 85°C for 5 min.RNA was extracted from the isolated blood using the TRIzol method , following the manufacturer’s protocols. cDNA preparation was performed in a 20-μl mixture condition using HifarTBX2, GATA3, Foxp3, RORC, BCL6, and SPI1 genes and housekeeping GAPDH as a reference gene were retrieved from the GenBank database with their accession numbers as illustrated in C 1. For qRT-PCR expression profiles, primer pairs of each target gene were designed by Premier 6.0 software, where their length ranged from 18 to 25 bp with 55%–65% of GC content and different annealing temperatures  as follows: 1 μl of cDNA, 10 μl of Master Mix, 0.4 μl of forward primer, 0.4 μl of reverse primer, and 8.2 μl of sterile RNase-free water. The amplification conditions started with initial denaturation at 95°C for 2 min, followed by 40 cycles of denaturation at 95°C for 10 s, 57°C for 30 s at annealing temperature, and the melting curve stage at 95°C for 15 s, 55°C for 15 s, followed by 95°C for 15 s.CDS sequences of eratures Table 1.According to the manufacturer’s instructions (Shanghai MLBIO Biotechnology Co. Ltd), an ELISA test was performed to measure nine cytokine levels—IFN-γ, TNF-α, IL4, IL10, TGF-β1 IL17A, IL6, IL9, and IL21—for the control and experiment group. Concentrations of plasma cytokines were calculated by using ELISA Calc v 0.1 software.Rabbits were euthanized on days 5, 15, and 23 PI. Partial left lobes of the livers from all rabbits were fixed in 10% neutral buffered formalin and embedded in paraffin for histological examination.The total liver weight was determined by electronic weighing, then 10 g of the left liver lobe was ground, dissolved in 50 ml of physiological saline, and filtered with an 80-mesh filter screen. Counts were performed with a hemocytometer.TBX2, GATA3, Foxp3, RORC, BCL6, and SPI1, as well as plasma cytokine levels of IFN-γ, TNF-α, IL4, IL6, IL10, IL9, IL17, IL21, and TGF-β1. Differences were considered significantly different when p < 0.05. Regression and correlation analyses were performed to detect the relationships between each mRNA transcriptional factor and every cytokine individually. Another set of regression and correlation was performed to determine the relationships between different mRNA transcriptional factors and the relationships between transcriptional factors and oocyst data, all at three time points . |r| values ≥ 0.8 were considered statistically significant.The general linear model procedure in SPSS 22.0 version was used to analyze mRNA expression levels of E. stiedai infection on rabbit livers was investigated using stained paraffin-embedded tissues. In control group, as shown in E. stiedai cells was observed , whereas TBX2 mRNA (p < 0.05) expression and IFN-γ (p > 0.05) were elevated on day 23 PI  without any effect on GATA3 mRNA. On day 23 PI, the infected group showed a decrease in GATA3 (p < 0.01), and IL4 was not significantly different (p > 0.05)  3C.RORC mRNA (p < 0.001) and IL17A (p < 0.01) in serum were significantly increased on day 5 PI, but there was no statistical difference in the IL6 serum levels. On day 15 PI, RORC mRNA (p < 0.001), IL17A (p < 0.05), and IL6 (p < 0.01) were significantly upregulated. On day 23 PI, RORC mRNA (p < 0.01) and IL17A (p < 0.05) oppositely decreased. Interestingly, IL6 serum levels were upregulated during the three periods in the experiment groups  on day 5 PI, and IL10 and TGF-β1 were also significantly increased (p < 0.01), compared with normal control. Interestingly, on day 15 PI, the level of Foxp3 was significantly decreased (p < 0.001); however, IL10 and TGF-β1 were still significantly increased (p < 0.01) compared with normal control. On day 23 PI, the levels of Foxp3 and relative cytokines (IL10 and TGF-β1) in serum were significantly decreased (p < 0.05) (The level of  < 0.05) 3F, G.SPI1 mRNA was significantly increased (p < 0.01); however, there was no statistical difference in IL9 (p > 0.05). On day 15 PI, SPI1 mRNA (p < 0.001) and IL9 (p < 0.01) were significantly elevated. On day 23 PI, the level of SPI1 mRNA was significantly decreased (p < 0.05), but there was no statistical difference in the concentration of IL9 between the normal control and infected groups  on day 5 PI. This influence was relatively stable on day 15 PI . On day 23 PI, the levels of BCL6 mRNA and IL21 showed no statistical difference between the normal control and infected group (p > 0.05) (r ≥ 0.8) were found between RORC and IL17A, SPI1 and IL9, and BCL6 and IL21 in the experiment groups throughout the whole period of infection as shown in TBX2 was positively correlated to TNF-α on days 5 and 15 PI. Cross-correlations were detected between transcriptional factors and cytokines of other T helper subsets, where TBX2 was negatively correlated to IL10 on day 5 PI. GATA3 was positively correlated to IL6 during the whole period of infection. GATA3 was negatively correlated to IL4 on day 5 PI. Foxp3 reported a positive correlation with IL4 and TGF-β1 (r ≥ 0.8) on day 5 PI, and RORC negatively correlated to TGF-β1 on days 15 and 23 PI.There are relationships between Th1/Th2/Th17/Treg/Th9/Th21 transcriptional factors with their cytokine lineages, where positive correlations , GATA3 (Th2), RORC (Th17), Foxp3 (Treg), SPI1 (Th9), and BCL6 (Th21)—and analyzed the potential effects of their imbalance on disease outcomes in the rabbit. Moreover, E. stiedai infections provide an excellent model to study the roles of Th cell transcriptional factors. In the early stage of the infection with E. stiedai (on day 5 PI), Th1 dominated at both transcriptional and protein levels. We also found a Treg response in the late stage of infection. Furthermore, our study also showed that Th17, Th9, and Th21 were involved in E. stiedai infection. However, helminth infection is characterized by Th2-dominated immune responses, which may contribute to chronic infection and the host’s inability to clear the parasites  depends on IRF8 and SPI1 in vivo, which is essential for developing germinal center B cells, and SPI1 acts as a regulator of BCL6 expression during GC B-cell development  were significantly decreased in the late stage, which suggests that they were suppressed.In addition, many cross-modulations were observed; TBX2 suppressed day 5 PI . Cross-mory site . On dayselopment . GATA3 poduction , but a soduction . A studyoduction . Treg onoduction . In geneoduction . Our stuE. stiedai made liver lesions severe , and TGF-β1.In the experiment group, liver weight increased, and the significant proliferation of eggs of s severe Table 3.E. stiedai-infected rabbits. Additional studies are needed to elucidate the detailed roles of these Th cells in the pathogenesis of E. stiedai.Our findings provide the first evidence of dynamic changes in Th1/Th2/Th17/Treg/Th9/Th21 and confirm an imbalance of these cell populations in The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by the Institutional Animal Care Committee of Sichuan Animal Science Academy.Conceptualization: X-DC, R-MK, and Y-GY. Methodology: X-DC. Software: LX, C-JM, and X-JW. Validation: J-FY, YW, and JX. Formal analysis: X-DC and YC. Resources: Y-GY. Data curation: R-MK and Y-GY. Writing—original draft preparation: X-DC. Writing—review and editing: Y-GY. Visualization: X-DC. Supervision: Y-GY. Project administration: R-MK and Y-GY. All authors contributed to the article and approved the submitted version.