The EPDM/flax composites exhibited higher values of dielectric constant, dielectric loss and conductivity as compared to a pristine EPDM sample. Using thermogravimetric analysis (TG) coupled with Fourier transform infrared spectroscopy (FTIR) and mass spectrometry (MS) (TG/FTIR/MS system), the degradation products can be identified. The water uptake increased as the flax fiber level increased in composites. The water uptake tests of irradiated composites showed that the highest water content was obtained for a flax fiber level of 20 phr.This paper deals with the dielectric and sorption properties of some flax fiber-reinforced ethylene-propylene-diene monomer (EPDM) composites containing different fiber loadings as well as their behavior after exposure to different doses of electron beam irradiation. Three relaxation processes were evinced, a weak relaxation β at sub-T Ethylene propylene diene terpolymer (EPDM) is a widely investigated commercially available elastomer. EPDM consists of ethylene and propylene units which provide a chemically saturated and stable backbone and the third monomer is a unconjugated diene. The polymerization reaction can be performed in a controlled way in order to keep the saturated backbone and to place the reactive unsaturation in a side chain where it is available for chemical modifications or vulcanization ,2. Due tEPDM rubber has several uses in the automotive industry , electrical and construction industries, aerospace or nuclear technology. EPDM is also utilized as a dielectric material for cable and wire insulation, especially cables in nuclear power plants which are subjected to thermal and radiation attacks during their utilization, and as an insulator matrix in rocket combustion chambers due to its excellent mechanical, chemical and electrical characteristics ,8,9,10.EPDM rubber can be vulcanized by peroxides or using a sulfur-accelerated system, which it is the most often used method for this material ,4. In coNatural fibers can represent an alternative reinforcement for EPDM composites due to their advantages over carbon and glass fibers such as low production costs, less health risks, biodegradability, etc. ,22. FromThe present work focused on the dielectric properties of some EPDM/flax composites in terms of dielectric constant, dielectric loss and conductivity. The effect of electron beam irradiation on the dielectric characteristics was analyzed with the respect to different flax loadings. The dielectric relaxation of the EPDM/flax composites was also discussed. The thermal stability of EPDM composites determined using the TG/FTIR/MS techniques and the water absorption properties were reported.1+4 at 120 °C, with an ethylene content of 70% and a density of 0.88 g/cm3 was supplied by Dow Chemical . The flax fibers were cut to a length of 3 mm. The features of the working materials presented in Ethylene propylene diene monomer Nordel 4760), with Mooney viscosity of 70 ML760, with2O3 crucibles, Al2O3 being the reference material. The released gases from the TGA instrument were analyzed using an online connected FTIR spectrometer  equipped with an external module TGA-IR and an Aeolos QMS 403C mass spectrometer (Netzsch). The IR absorption spectra were obtained over the wavenumber range of 600–4000 cm−1 at a resolution of 4 cm−1. The FTIR transfer line of the evolved gases was made from polytetrafluoroethylene heated at 190 °C. The acquisition of FTIR spectra in 3D format was performed with the OPUS 6.5 software. The gas transfer line to mass spectrometer was made from a quartz capillary heated at 240 °C. QMS 403C spectrometer works at 10−5 mbar vacuum and electron impact ionization energy of 70 eV. The data acquisition was achieved in the range m/z of 1-300, measurement time of 0.5 s.Thermogravimetric analysis of EPDM composites was conducted in dynamic conditions on a STA 449F1 Jupiter instrument  in nitrogen atmosphere, at a heating rate of 10°/min in the temperature range of 30–700 °C. Samples of 10–15 mg were placed in AlAll sorption measurements were realized with an IGAsorp dynamic sorption analyzer  equipped with an ultrasensitive microbalance taking into account the modifications in the sample mass against the temperature and relative humidity. Before sorption experiment, each sample was dried in nitrogen flow (250 mL/min) until the weight was in equilibrium at relative humidity (RH) < 1%. The measurements were made at 25 °C in a RH range of 0–90%, using humidity steps of 10%, each step having a pre-established equilibrium time between 5 and 10 min.6 Hz in the temperature range from −120 °C to 120 °C. The measured geometry was a plate-capacitor formed by a disc shaped sample having 1 mm thickness and placed between two round gold-plated flat electrodes of 20 mm diameter of the test capacitor. All the samples were gold-coated on their both surfaces, in order to assure a good electrical contact between the sample surfaces and the electrodes used to carry the dielectric measurements.The measurement of dielectric properties of the EPDM/flax composites was carried out with a broadband dielectric spectrometer . The spectrometer was equipped with an Alpha–A high frequency analyzer. The determinations were done over a frequency window of 1–10Irradiation of the EPDM/flax composites was achieved on an ALIN10 electron beam accelerator at room temperature as described in . SpecimeIn the present study, two main aspects were followed by the dielectric technique: the modification of molecular mobility and the variation of the electrical resistivity for nonirradiated and electron beam irradiated EPDM composites as compared to the initial EPDM sample. The mobility in composites can be firstly affected by the physical immobilization of polymer chains imposed by the flax fibers and then, by the crosslinking process derived from the electron beam irradiation. It is known, that in the EPDM/carbon black composites, NMR relaxation experiments proved unambiguously the immobilization of EPDM chain fragments on the surface of carbon black filler . In thisThe frequency dependences of the dielectric constant, ε′, dielectric loss, ε″ and conductivity, σ, at room temperature for EPDM sample without flax (E0) and EPDM/flax composites are given in −11 S/cm, respectively), revealing the insulating character of these composites. In the low frequency range, the dielectric constant and dielectric loss have the highest values taking into account the free charges in composites [As follows from mposites . This efmposites ,32. It iIn 2 groups which may have attached some more polar impurities, similar to the low temperature γ-relaxation observed around −100 °C in polyurethanes [rethanes ,33.The variation of dielectric constant and dielectric loss against temperature with respect to frequency is shown in With further increasing temperature, two dielectric relaxations were observed. They are correlated with the glass transition of EPDM chains located in the amorphous phase and among the crystallites, being designed as α and α′, respectively. For EF10 composite and the irradiated EF10-600 sample, the α and α′ dipolar relaxations are strongly overlapped in both the ε′ and ε″ representations. In addition, for composite-type materials, a frequency independent process due to the melting of crystallites is visible with further increases in temperature. This physical process is detected between 40 and 45 °C for the nonirradiated EF10 composite and 45–50 °C for the irradiated composite EF10-600. The melting process was previously observed by DSC thermograms between 34 and 46 °C for EF10 and 40–56 °C for irradiated samples, respectively,  confirmiThe 3D representations of the dielectric loss as a function of frequency and temperature are rpresented in HN is the HN relaxation time of the dipolar process, αHN and βHN are the shape-related parameters of the dielectric peak [max, was evaluated with the following expression:In this section, the detailed analyses of the segmental relaxation will be performed, following the interaction between polymer chains and flax fibers. For this reason, the ε″ (f) dependencies were processed based on the Havriliak-Negami (HN) relation:ric peak . Further0 is a prefactor, k is the Boltzmann’s constant, T is the absolute temperature and Tv is the so-called Vogel temperature.The main relaxation peaks, α and α′, are related to the cooperative reorientation motions of the main segments. As previously noticed, the α-relaxation exhibits as a dielectric peak with reduced intensity. It is strongly overlapped by the α′-relaxation and, consequently, its curvature cannot be exactly identified and processed. On the other hand, α′-relaxation appears as a well-shaped dielectric peak with high intensity. This signal will be further processed for simple E0 polymer, EF5, EF10 and EF15 nonirradiated composites and also for E0-75 and E0-300 irradiated composite samples. For other samples, we believe that the dielectric peak specific to α′-relaxation is suppressed by the crosslinking process. The activation energy of the segmental α′-relaxation, B, was finally determined with the Vogel-Fulcher-Tammann (VFT) expression:5 Hz) related to the peak of α-relaxation. A supplementary term for the MWS process is limited at low frequencies. The dielectric peak of α′ relaxation was found asymmetric (βHN ~0.7–0.8) and narrow (αHN ~0.55–0.65). Closed values of βHN and αHN were also found for EF5, EF10 and EF15 nonirradiated composites.g values of E0 and composites are comparable.The activation energy of E0 polymers and the corresponding composites are quite close b inset, 4 Hz) and a small term for the dielectric signal of MWS are enclosed. The HN shape parameters for α′-relaxation of E0-300 sample were obtained as βHN ~0.75 and αHN ~0.65 (on average over temperature), being similar with that of nonirradiated composites. However, according to g for e-beam irradiated composites increased with the irradiation dose due to the reduced molecular mobility provided by crosslinking [The ε″(f) dependencies of E0-300 sample at temperatures between 10 and 40 °C are displayed in −1, respectively. The absorption band around 3200 cm−1 (ice band) is characteristic of the MCT detector (cooled with liquid nitrogen) of the TG-IR external module. The absorption bands located at 3749–3566 and 1295–1375 cm−1 (ν OH) can be assigned to water vapors and the bands around 2360–2300 and 668 cm−1 to carbon dioxide. The specific signals of the CH, CH2 and CH3 groups (νCH stretching vibrations) of aliphatic saturated derivatives correspond to the absorption bands around 2925–2853, 1375–1294, 712–720 cm−1 and aliphatic unsaturated derivatives (νC=C) show absorption bands in the range of 1646–1688 cm−1 [−1, νCH at 899 cm−1 and νC=O groups specifically of furan ring at 1124–1067 cm−1. The aromatic derivatives show absorption bands at 3078–3010 cm−1 (ν CH), ring vibrations, νC=C, at 1456 cm−1 and substitution bands, γCH, at 952, 821 cm−1.In order to study the thermal stability and the degradation mechanism of the EPDM and corresponding composites, TG-FTIR analysis was applied. The low molecular weight volatile compounds arising during thermal degradation were identified by TG/FTIR/MS simultaneous analysis. The 3D FTIR diagrams resulting from the thermal decomposition of the EPDM samples are presented in m/z values at the temperature (Tmax) corresponding to the maximum amount of released gases are given in m/z signals given in 2O+) m/z = 18, 17, 16; carbon dioxide (CO2+) m/z = 44, 28, 16, 12, 45; alkane and alkene derivatives such as propylene (C3H6+) m/z = 42, 41, 39, 27, 40, 38, 37, 26, 15); propane (C3H8+) m/z = 44, 29, 27,43, 39, 41, 26, 15, 42; butane (C4H10+) m/z = 58, 43, 29, 27, 28, 41, 39, 42, 15, 26; hexene (C6H12+) m/z = 84, 56, 41, 42, 55, 43, 27, 39, 69, 29; heptene (C7H14+) m/z = 98, 56, 41, 55, 29, 42, 70, 69, 57, 39, 27; norbornene (C7H10+) m/z = 94, 66, 39, 79, 67, 77, 65, 27, 40, 41; 5-ethylidene 2-norbornene (C9H12+) m/z = 120, 66, 91,105, 78, 39, 77, 79, 65, 92; trimethylcyclohexane (C9H18+) m/z = 126, 69, 111, 55, 41, 39, 56, 70, 42, 112; aromatic derivatives such as, benzene (C6H6+) m/z 78, 77, 51, 50, 52, 39, 70, 76, 38 and toluene (C7H8+) m/z = 92, 91, 65, 39, 63, 51, 93, 50, 89, 62.MS signals represented by 4H8+) or aromatic (C6H7+) derivatives can appear (m/z = 120 (4H4O+) m/z = 68, 39, 38, 40, 29, 37, 42, 60; furan aldehyde (C5H4O2+) m/z = 96, 95, 39, 38, 29, 37, 67, 40, 97, 42; 2(5H)-furanone (C444O2+) m/z = 84, 55, 27, 26, 54, 29, 39, 38, 27; 5-methyl-2-furaldehyde (C6H6O2+) m/z = 110, 109, 53, 27, 39, 51, 81, 43, 51, 111 [In the MS spectra of EPDM/flax composite (EF20) an additional process of thermal degradation occurring up to 400 °C was found. In this range water, carbon dioxide as well as ionic fragments of some aliphatic MttM/M∞ < 0.5 and sufficiently long times, tM/M∞ > 0.5, one can write that:Taking into account the approximation of the first terms in Equations (4) and (5), at sufficiently short times, W is the weight of adsorbed water, mW is the weight of water forming a monolayer, C represents the sorption constant and p/po is the relative humidity. The BET model allows one to calculate the surface area in a range of activities from 0.00 to 0.35. The BET data are summarized in Using the above diffusion models the results regarding the water uptake for EPDM/flax fiber composites are presented in rpm is the average pore size, A denotes the BET surface area, n is the percentage uptake, aρ is the solvent phase density.Assuming cylindrical pore geometry, the average pore size was estimated using the relation (6)  Table 2Table 2:(As was expected, the water absorption in nonirradiated EPDM composites increases as the level of flax fiber increased  attainin3 Hz. The dielectric constant and dielectric loss increased with increasing flax fiber loading due to the increase of the number of polar groups in the composites leading to a higher orientation polarization. Overall, the EPDM/flax composites revealed higher values of dielectric parameters as compared to a pristine sample. The dielectric relaxation behavior of EPDM/flax composites exhibits several relaxation processes as the temperature increases, namely β-relaxation which corresponds to local motions occurring at lower temperatures and α, α′-relaxations associated to the cooperative reorientation motions of the polymer segments. The activation energy of the α′-relaxation was estimated using Vogel-Fulcher-Tammann relation having higher values for electron beam-irradiated samples. The water uptake of EPDM composites was influenced by the flax level and the irradiation dose. The simultaneous analysis of the thermal degradation of the EPDM/flax composites by TG/FTIR/MS technique showed details about the polymer decomposition process. The main gaseous products evolved during the composite degradation were determined. By tuning composition and crosslink density the desired properties of EPDM/flax fiber composites can be achieved in order to be used as polymeric insulators.The dielectric parameters such as dielectric constant and dielectric loss monotonically decreased with the increase of frequency, but they become independent as the frequency increased above 10


Cellulase synthesized by fungi can environment-friendly and sustainably degrades cellulose to fermentable sugars for producing cellulosic biofuels, biobased medicine and fine chemicals. Great efforts have been made to study the regulation mechanism of cellulase biosynthesis in fungi with the focus on the carbon sources, while little attention has been paid to the impact and regulation mechanism of nitrogen sources on cellulase production.Trichoderma reesei, followed by yeast extract, urea, tryptone, ammonium sulfate and l-glutamate. Cellulase production, cell growth and sporulation in T. reesei RUT-C30 grown on cellulose were all inhibited with the addition of glutamine (a preferred nitrogen source) with no change for mycelium morphology. This inhibition effect was attributed to both l-glutamine itself and the nitrogen excess induced by its presence. In agreement with the reduced cellulase production, the mRNA levels of 44 genes related to the cellulase production were decreased severely in the presence of glutamine. The transcriptional levels of genes involved in other nitrogen transport, ribosomal biogenesis and glutamine biosynthesis were decreased notably by glutamine, while the expression of genes relevant to glutamate biosynthesis, amino acid catabolism, and glutamine catabolism were increased noticeably. Moreover, the transcriptional level of cellulose signaling related proteins ooc1 and ooc2, and the cellular receptor of rapamycin trFKBP12 was increased remarkably, whose deletion exacerbated the cellulase depression influence of glutamine.Glutamine displayed the strongest inhibition effect on cellulase biosynthesis in T. reesei RUT-C30. More importantly, the presence of glutamine notably impacted the transport and metabolism of nitrogen. Genes ooc1, ooc2, and trFKBP12 are associated with the cellulase repression impact of glutamine. These findings advance our understanding of nitrogen regulation of cellulase production in filamentous fungi, which would aid in the rational design of strains and fermentation strategies for cellulase production in industry.Glutamine may well be the metabolite effector in nitrogen repression of cellulase synthesis, like the role of glucose plays in carbon catabolite repression. Glutamine under excess nitrogen condition repressed cellulase biosynthesis significantly as well as cell growth and sporulation in The online version contains supplementary material available at 10.1186/s13068-021-02046-1. Penicillium occitanis [Penicillium funiculosum [Gibberella fujikuroi [Fusarium graminearum [Aspergillus flavus [In nature, organisms sense nutrient availability in the surrounding environment and adjust their metabolism for optimal growth, development, and reproduction . Carbon ccitanis  or Peniciculosum , gibbereujikuroi , deoxyniminearum , and afls flavus . Study hT. reesei, Aspergillus nidulans, and P. funiculosum. In T. reesei, deletion of the global nitrogen regulator Are1 decreased cellulase production when using (NH4)2SO4 as the nitrogen source, demonstrating a role of Are1 in the regulation of cellulase production [A. nidulans, cellulase production is impacted by both carbon and nitrogen source, and is under the regulation of the carbon regulators CreA, CreB, and CreC, as well as the nitrogen regulator AreA, either directly or indirectly [P. funiculosum by evaluating various nitrogen sources including ammonium sulfate, urea, yeast extract, and peptone [T. reesei [T. reesei can consume a variety of nitrogen-containing compounds with little change in cellulase production, while nitrogen limitation inhibited cell growth and cellulase biosynthesis [Nitrogen metabolism plays an important role in cellulase production in filamentous fungi like oduction . In A. ndirectly . Meanwhi peptone . Suitabl. reesei , 17. T. ynthesis –25, lessT. reesei RUT-C30 grown under cellulase-producing condition was investigated in terms of cellulase synthesis, cell growth, sporulation ability, and morphology. The molecular mechanism behind glutamine-repressed cellulase production was explored by comparative transcriptional profiling and the knockout of genes ooc1, ooc2, and trFKBP12.To investigate the nitrogen regulation of cellulase production in fungi, we evaluated the influence of various nitrogen sources on cellulase production under excess complex nitrogen condition, finding that glutamine exhibited the strongest inhibition effect on cellulase biosynthesis. Then the effect of glutamine on Trichoderma minimal media (TMM) on cellulase production, including glutamine, glutamate, ammonium sulfate, yeast extract, tryptone, and urea  into TMM + 2% cellulose cellulase production of T. reesei  with coverage of 93.98–94.51%. A total of 10,048 unique transcripts were detected  were obtained, of which 699 were upregulated and 493 were downregulated  functional enrichment analysis of these DEGs showed that the most enriched molecular function was “catalytic activity”, which includes four enriched subcategories “hydrolase activity”, “oxidoreductase activity”, “β-glucosidase activity”, and “xylanase activity” Fig. . The othThe top 29 enriched KEGG pathways were obtained by KEGG pathway enrichment analysis of DEGs cellulase genes including 2 cellobiohydrolases (CEL7A and CEL6A), 7 endoglucanases , 6 β-glucosidases , 6 xylanases , and two β-xylosidases (BXL1 and BXL) were all reduced markedly, matching well with the decreased (hemi)cellulase activities as observed above. Genes encoding the auxiliary proteins like swollenin [cel3g, M419DRAFT_93487 and M419DRAFT_6566 were increased with the addition of glutamine. However, the reason behind this phenomenon is unknown as these three genes are lack of study, which is worthy of research in future study. This suggests that cellulase productions are subject to nitrogen regulation at the transcriptional level.A total of 86 genes were known or predicted to be associated with (hemi)cellulase biosynthesis in wollenin , and thewollenin , which hwollenin , Ace3 [2wollenin , Clr2 [3wollenin , and Vibwollenin , 32, andwollenin , 34. We 15 DEGs are involved in ribosome biogenesis, all of which were downregulated except gene M419DRAFT_104236  to glutamate. Then glutamine synthase (GS) uses glutamate and ammonia to synthesize glutamine. Both these genes were significantly downregulated as well as the ammonium permease (MepC) , urea (M419DRAFT_84192), oligopeptide (M419DRAFT_84192), arginine (M419DRAFT_91864) were dramatically downregulated, while transporters for amino acids and 4-aminobutanoic acid (GABA), were significantly upregulated. The upregulated expression of amino acid transporters might be involved in the uptake of glutamine. Gene encoding GABA transaminase (M419DRAFT_87303) was also increased. GABA is an intermediate metabolite in the GABA shunt, a metabolic pathway that bypasses two enzymatic steps of the tricarboxylic acid (TCA) cycle to produce succinate from α-ketoglutarate via glutamate  catalyzes the conversion of α-ketoglutarate (α-KG) and ammonia (NHpC) Fig. , indicatAs the GS/GOGAT cycle and the GABA shunt link primary nitrogen and carbon metabolism , the priooc1 and ooc2 in the responsiveness of T. reesei to glutamine. Genes ooc1 and ooc2 were deleted separately in T. reesei KU70, leading to recombinant strain Δooc1 and Δooc2, respectively. Strain KU70, a ku70-deleted derivative of RUT-C30, was utilized as the parent strain for its high efficiency of gene targeting [ku70 renders the resistance of T. reesei RUT-C30 to glutamine. Further deletion of ooc1 and ooc2 individually brought back the cellulase inhibition effect of glutamine in strain KU70   was incrargeting . InteresT. reesei. Unexpectedly, transcriptional analysis showed that neither TOR nor the components of TORC1 and TORC2 were changed significantly after the treatment of glutamine for 24 h -fold with 2.92 g/L glutamine. To see whether trFKBP12 was involved in the negative impact of glutamine on cellulase production, the trFKBP12 deletion strain ΔtrFKBP12 engineered in our previous study [ooc1 and ooc2, the knockout of trFKBP12 exacerbated the cellulase depression influence of glutamine  kinase signaling pathway was a central signaling hub integrating cell growth and metabolism –39. It c4 h Fig. A. Furthe4 h Fig. B. For ge4 h Fig. C. Rrn3 wrevisiae . M419DRArevisiae  were alsine Fig. D, suggesT. reesei RUT-C30. Glutamate displayed the weakest inhibition effect on cellulase production, followed by ammonium sulfate, tryptone, urea, yeast extract and glutamine in ascending order. The cellulase inhibition was found regardless of nitrogen sources, while the inhibition degree was dependent on nitrogen types. This suggests that the cellulase inhibition was due to both the nitrogen excess induced by the addition of more nitrogen sources and the administered nitrogen source itself. Although the nitrogen content of different nitrogen sources varies greatly at 2.92 g/L, 40.0 mM for glutamine, 97.2 mM for urea, 44.2 mM for ammonium sulfate, and 19.8 mM for glutamate, there is no notable correlation between the nitrogen content and cellulase inhibition. For example, the inhibition effects of 2.92 g/L glutamine and ammonium sulfate on cellulase production differed greatly, although they contained a similar nitrogen content. Despite of the most severe inhibition for glutamine on cellulase production, the nitrogen content of glutamine was between those of glutamate (19.8 mM) and urea (97.2 mM). Clearly, the cellulase inhibition effect was related to the nitrogen type rather than the nitrogen content. Glutamine possessed the strongest cellulase repression effect among all the tested nitrogen sources. Although both ammonium sulfate and glutamine are preferred nitrogen sources, glutamine displayed much stronger cellulase repression than that of ammonium sulfate, indicating that ammonium sulfate is a more suitable nitrogen source for cellulase production in T. reesei than glutamine. Meanwhile, glutamine and glutamate displayed a huge difference in the inhibition degree on cellulase production, though they can be converted to each other through the GS/GOGAT cycle in cells. It seems that glutamine may well be the metabolite effector in nitrogen repression of cellulase synthesis, similar to the role of glucose plays in CCR.To varying degrees, the supply of different nitrogen sources at equal amount to TMM culture medium inhibited cellulase production in T. reesei might be utilized in preference to other nitrogen sources. Therefore, the presence of glutamine caused a significant change on nitrogen transport and metabolism in T. reesei. We can see the supply of glutamine shut down the transport of other nitrogen sources including ammonium, urea, oligopeptide (M419DRAFT_84192), and arginine, but enhanced its transport through the upregulation of amino acid transporters  [pkac1 (protein kinase) [cre1 [Trichoderma harzianum T34, ooc1 was induced during its interactions with Pythium ultimum when nox1 was overexpressed [T. reesei RUT-C30 increased the expression of genes ooc1 and ooc2 to antagonize the cellulase-depression effect of glutamine. All these demonstrate that gene ooc1 and ooc2 in T. reesei RUT-C30 were highly responsive to various signals in the environment.Genes sponsive . It seemprotein) , Δpkac1  kinase)  or Δcre1e) [cre1  strains xpressed . In thistrFKBP12 was increased remarkably. This was similar to what we found previously that genes related to TOR signaling pathways were not changed significantly but trFKBP12 was significantly upregulated by rapamycin in T. reesei grown on cellulose condition [trFKBP12, and the downregulated expression of transcription initiation factor Rrn3, stress-responding transcription factors Msn2 and Msn4, as well as the primary amino acid receptor Ssy1. In addition, the deletion of trFKBP12 reinforced the inhibition of glutamine on cellulase synthesis, showing that gene trFKBP12 is related to the glutamine-inhibited cellulase production in T. reesei.The TOR kinase, a central regulator of nutrient response, plays an important role in cell growth and metabolism and could be activated by glutamine . Howeverondition . NeverthT. reesei as an example, we first studied the effect of glutamine on cellulase-producing fungi under cellulose condition, including cellulase production, cell growth, sporulation, mycelium morphology and transcriptome. Under nitrogen excess condition, glutamine displayed the strongest inhibition effect on cellulase production, followed by ammonium sulfate, tryptone, urea, yeast extract and glutamine in descending order. Cellulase production was declined and the growth as well as sporulation were impaired with the addition of glutamine, while the mycelium morphology was not changed. Further RNA-seq analysis showed that genes associated with the cellulase production were decreased severely at the mRNA level in the presence of glutamine. Moreover, glutamine exerted a remarkable change on the transcriptional levels of genes related to nitrogen metabolism, such as the decreased expression of genes taking part in non-glutamine nitrogen transport, ribosomal biogenesis and glutamine biosynthesis, and the enhanced expression of genes associated with glutamate biosynthesis, amino acid catabolism, and glutamine catabolism. Genes ooc1, ooc2 and trFKBP12 are associated with the cellulase repression impact of glutamine in T. reesei. All together, we found that glutamine is probably the metabolite effector in nitrogen repression of cellulase synthesis by significantly changing nitrogen transport and metabolism, which is facilitated by genes ooc1, ooc2, and trFKBP12. These findings provide a comprehensive understanding of the regulatory mechanism of nitrogen in cellulase production in filamentous fungi, which would help in establishing optimal fermentation strategies for better cellulase production in industry.Using Escherichia coli DH5α was used as the cloning host for plasmid construction. Agrobacterium tumefaciens AGL-1 was used as a T-DNA donor for fungal transformation. T. reesei RUT-C30  was purchased from China Center of Industrial Culture Collection. T. reesei KU70, where ku70 was deleted in RUT-C30 [E. coli DH5α and A. tumefaciens AGL-1 were cultivated in Luria–Bertani (LB) with 220 rpm at 37 °C and 28 °C, respectively. T. reesei were grown on potato dextrose agar (PDA) plates for conidia production and in TMM [4)2SO4, 4.00 g/L; KH2PO4, 6.59 g/L; Maleic acid, 11.6 g/L; FeSO4 * 7H2O, 0.005 g/L; MnSO4 * H2O, 0.0016 g/L; ZnSO4 * 7H2O, 0.0014 g/L; CoCl2 * 6H2O, 0.002 g/L; MgSO4, 0.60 g/L; CaCl2, 0.60 g/L; Tween 80, 0.186 ml/L [ RUT-C30 , was prod in TMM  with 2% 186 ml/L . When gl7 /mL) conidia of T. reesei were inoculated into 10 mL sabouraud dextrose broth (SDB) and cultured at 28 °C with 200 rpm for 2 days. 5 mL pre-grown mycelia were inoculated into 50 mL TMM media (pH 6) plus 2% cellulose with different concentrations of glutamine and incubated at 28 °C with 200 rpm for 7 days. The stock solution of glutamine (100 g/L) was prepared in water. Samples were taken at indicated timepoints for (hemi)cellulase activity assay, confocal observation, biomass dry weight measurement, and RNA-seq analysis. The samples of T. reesei culture collected above were centrifuged at 8000 rpm for 30 min to remove the cell pellets, leading to the supernatants for (hemi)cellulase activities assay as described in our previous research [Five percent  with a 100 × oil immersion objective. The spores were counted by a hemocytometer under a confocal microscope SP8 with a 20 × oil immersion objective. The relative mRNA level was normalized using the housekeeping gene sar1 [(Hemi)cellulase activity assay, confocal imaging, spore counting, RNA-seq analysis, and RT-PCR were performed as described in our previous research , 65. T. ene sar1 . All theooc1, ooc2, and ooc1 and ooc2 were separately amplified by PCR using genomic DNA of T. reesei KU70 as a template, and cloned into plasmid pXBthg at XhoI and at BamHI using ClonExpress™ II One Step Cloning Kit , leading to plasmids pXBthg-ooc1, pXBthg-ooc2, and pXBthg-ooc12, respectively. Genes ooc1 and ooc2 can be deleted simultaneously as they are next to each other in T. reesei genome. The resulting plasmids pXBthg-ooc1, pXBthg-ooc2, and pXBthg-ooc12 were individually transformed into T. reesei KU70 separately by the Agrobacterium tumefaciens-mediated transformation (AMT) method using hygromycin B as a marker [The upstream and downstream sequences (~ 1500 bp) of gene a marker , yieldinAdditional file 1: Table S1. The comparative transcriptome in T. reesei RUT-C30 cultured in TMM with/without the addition of glutamine.Additional file 2: Table S2. Total DEGs in T. reesei RUT-C30 cultured in TMM with the addition of glutamine.Additional file 3: Table S3. Genes involved in (hemi)cellulase production in T. reesei RUT-C30.Additional file 4: Table S4. DEGs related to “ribosome biogenesis” in T. reesei RUT-C30 cultured in TMM with the addition of glutamine.Additional file 5: Table S5. DEGs involved in amino acids in T. reesei RUT-C30 cultured in TMM with the addition of glutamine.Additional file 6: Table S6. Genes involved in TOR signal pathways in T. reesei RUT-C30.Additional file 7: Table S7. Genes involved in nitrogen and carbon catabolite repression in T. reesei RUT-C30.Additional file 8: Table S8. Primers for gene cloning, PCR confirmation and qPCR.Additional file 9: Figure S1. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of DEGs. The y axis represents the name of the most enriched pathways. Figure S2. PCR confirmation of recombinant T. reesei strains △ooc1, △ooc2, and △ooc12.


Anvillea garcinii is a medicinal plant used in the Arab region for intestinal diseases, lung and liver diseases, digestive problems, and as an antidiabetic agent. Repeated chromatographic purifications of A. garcinii leaves led to the isolation of three undescribed guaiane sesquiterpene derivatives, named garcinamines F–H, characterized by the presence of an amino acid unit, along with five known sesquiterpene lactones (garcinamines B–E and 9β-hydroxyparthenolide). The structures of the new compounds were established using spectroscopic (1D and 2D NMR) and spectrometric methods (ESIMS). Garcinamine H possesses a double bond at the Δ1,10 position, a structural feature rarely reported in guaianolide-type sesquiterpenes. The antiproliferative activity of the isolated sesquiterpenes was screened against three different cancer cell lines, and 9β-hydroxyparthenolide and garcinamines C and D displayed significant effects against lung carcinoma (A549), colon carcinoma (LoVo), and breast carcinoma (MCF7) cell lines. Cancer is the second-most life-threatening disease, with a worldwide incidence leading to nearly 9.5 million deaths and 18 million cases in 2018, with an expected doubling of cases and deaths by 2050 . Cancer Anvillea garcinii (Burm.f.) DC. (family Asteraceae) is one of the distinctive ethnomedicinal plants used in the Arabian Peninsula to cure many illnesses, such as cold, digestive problems, pulmonary infections, and liver diseases 25 D-27.2 ; (+) ESIMS m/z 380.2076 [M + H]+ ; 1H and13C NMR data, see Garcinamine F (2). Yellow gummy solid; [α]25D-22.5 ; (+) ESIMS m/z 380.2080 [M + H]+ ; 1H and13C NMR data, see Garcinamine G (3). Yellow gummy solid; [α]25D-29.3 ; (+) ESIMS m/z 380.2069 [M + H]+ ; 1H and13C NMR data, see Garcinamine H  to prepare stock solution (3 mg/mL). The serial dilutions of compounds (1–30 µM) were added to triplicate wells; mock-treated (0.1% DMSO) cells were used as negative control and doxorubicin was used as positive control. Cells were treated with the appropriate compounds for 48 h. At the end of the treatment period, 1 mL of 5 mg/mL MTT was added to each well, and the plates were further incubated at 37 °C in the presence of 5% CO2 for 2–4 h. The media were removed, 0.01 N HCl in isopropanol was added to each well, and the plates were placed in a shaker for 10 min. The absorbance was read at 570 nm using a microplate reader . The concentration at which cell viability was reduced by 50% (IC50) was calculated using Origin Pro 8.5 software.Cell proliferation assay was carried out using MTT. Three different types of human cancer cell lines  were used in this study. The cells were counted and plated in a 24-well cell culture plate with a density of 5 × 101–8 against breast (MCF-7), colon (LoVo), and lung (A549) cell lines were examined using MTT assay. The collected cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% heat-inactivated fetal bovine serum, HEPES buffer, 1% L-glutamine, and 50 µg/mL gentamycin, and then placed in 96-well plates at 50,000 cells/well. Compounds were dissolved in DMSO and the cells were treated with different concentrations of each compound for 48 h, and then 3--2,5 diphenyl tetrazolium bromide (MTT)  was added and cells were incubated for an additional 4 h at 37 °C. Thereafter, cells were lysed and dark blue crystals of the formazan product were dissolved with isopropanol and the absorbance intensity was measured at 570 nm using a microplate reader The cell survival percent was calculated as the mean absorbance of treated sample/mean absorbance of control × 100. The dose–response curve of the compounds was analyzed using OriginPro 8.5 software. Doxorubicin was used as a positive control.The antiproliferative effect of compounds 50 concentration and DMSO as a vehicle for 48 h. Thereafter, cells were harvested using trypsin and centrifuged and fixed in 70% ethanol for 4 h at 4 °C. Ethanol-fixed cells were pelleted, washed with ice-cold PBS, and re-suspended in a staining solution containing 50 μg/mL PI and 100 μg/mL RNase. After incubation in the dark for 30 min, the cells were analyzed by flow cytometry.Cellular DNA content was analyzed using propidium iodide staining by flow cytometry. In brief, A549 cells were seeded into a 6-well plate in 2 mL culture medium with an IC7 at IC50 values, A549 cells were collected, washed and resuspended in Annexin binding buffer. Thereafter, cell staining was performed with FITC and PI (5 µL of each), according to the manufacturer’s instructions . Flow cytometric analysis was performed immediately after 20 min of staining. Data acquisition and analysis were performed in a flow cytometer .The apoptotic cells were detected using flow cytometry with the Annexin V-FITC/PI double labeling method. Briefly, after 48 h of treatment with Anvillea garcinii allowed us to expand the knowledge on the sesquiterpenoid composition of this plant with the addition of three new guaianolide-proline adducts named garcinamines F–H. These compounds bear an evident biogenetic relationship with germacranolides, previously isolated from the same plant. All the isolated compounds were evaluated for their antiproliferative activity against three human carcinoma cell lines. Garcinamines C and D and 9β-hydroxyparthenolide exerted cytotoxic effects in a dose- and time-dependent manner. The mechanism of action of garcinamine D was investigated, revealing a marked pro-apoptotic effect with an accumulation of cells in the sub-G1 phase. These results further confirm that plants constitute an immense casket of chemodiversity that can be beneficially used to discover new hits for drug development.Phytochemical investigation of the aerial parts of


Aggressive human cervical cancer cells (CASKI) presented higher gene expression levels of EGFR, uPA, and TM compared to its less aggressive counterpart (C-33A cells). EGFR induces uPA expression in CASKI cells through both PI3K-Akt and MEK1/2-ERK1/2 downstream effectors, whereas TM expression induced by EGFR was dependent on PI3K/Akt signaling alone. uPA induced cell-morphology modifications and cell migration in an EGFR-dependent and -independent manner, respectively. Finally, treatment with cetuximab reduced in vivo CASKI xenografted-tumor growth in nude mice, and decreased intratumoral uPA expression, while TM expression was unaltered. In conclusion, we showed that EGFR signaling regulated expression of the fibrinolytic system component uPA in both in vitro and in vivo settings, while uPA also participated in cell-morphology modifications and migration in a human cervical cancer model.Epidermal growth factor receptor (EGFR) signaling and components of the fibrinolytic system, including urokinase-type plasminogen activator (uPA) and thrombomodulin (TM), have been implicated in tumor progression. In the present study, we employed cBioPortal platform ( Cervical cancer is the fourth most incident type of cancer among women, with 570,000 new estimated cases in 2018 .The epidermal growth factor receptor (EGFR) has been associated with cervical cancer progression ,5. EGFR A different group of receptors and secreted proteins that have been increasingly shown to participate in cancer invasion and metastasis is the fibrinolytic system. The fibrinolytic system is classically associated with the degradation of fibrin clots in the blood. However, the study performed by Malone and coworkers showed that cancer tissues from metastatic foci had increased fibrinolytic activity compared to primary non-metastatic tumor samples . The genEGFR signaling has been directly linked with components of the fibrinolytic system, for example, in breast cancer and glioblastoma. Jo et al.  showed tin vivo, we observed that treatment with an anti-EGFR antibody, cetuximab injected subcutaneously, reduced tumor growth, a process that was accompanied by a decreased intra-tumoral expression of uPA.It remains unanswered, however, if EGFR signaling is related somehow to components of the fibrinolytic system in cervical cancer. In the present work, we observed a positive expression correlation between EGFR and several fibrinolytic system elements, including uPA and TM on human cervical tumor samples, with prognostic significance. We also observed that EGFR signaling positively regulated expression of both uPA and TM in an aggressive cervical cancer cell line, and that uPA expression led to cell morphology alterations and increased migration. Finally, http://www.cbioportal.org/) (TM (USA), and analyzed by non-parametric Spearman correlation.Transcriptome data from 302 patients were collected from the “TCGA, Firehouse Legacy” cohort on the platform cBioPortal  ,24 betweTM. Mantel-Cox test was used for statistical analysis.From the cBioPortal platform, we established a cut-off of 1.5 standard deviations above the median of expression to separate groups of “overexpression” and “cases without alteration” of the cohort “TCGA, Firehouse Legacy”, from the 302 patients. After the establishment of the gene expression cut-off, we analyzed overall survival at the “survival” window on cBioPortal, extracted the data, and assembled the graphs with Graphpad2. CASKI cell line was originated from an intestinal metastasis of an epidermoid cervical carcinoma and was infected with multiple copies of HPV-16 . C-33A cells originated from a primary epidermoid cervical carcinoma, uninfected by HPV .Human cervical cancer cells CASKI (more aggressive) and C-33A (less aggressive) were cultivated in RPMI medium supplemented with 10% fetal bovine serum (FBS), and maintained in 37°C, 5% CO5 cells/well) onto 6-well plates, in RPMI medium supplemented with 10% FBS. The next day, the wells were washed with phosphate-buffered saline (PBS) and cells were starved (FBS-free medium) for 16 h. After starving, cells were treated with anti-EGFR antibody , PI3K inhibitor , or MEK inhibitor , for 1h. Then, cells were treated with EGF (50 ng/mL) for 1.5 h or 3 h, 37°C, 5% CO2. The wells were washed with PBS and 0.5-1 mL Trizol (Sigma-Aldrich) was added to each well. The extracts were transferred into microcentrifuge tubes and stored at -20°C, for posterior RNA isolation. From each sample, 1 µg of total RNA was reverse transcribed to cDNA. Next, quantitative PCR was performed on cDNA aliquots.Briefly, C-33A or CASKI were seeded , 0.5 µL TaqMan probe for human TM (Hs00264920_s1) or human GAPDH , 2 µL injection water, and 2.5 µL diluted cDNA (1:20 v/v) were added, totaling 10 µL per well of a 96-well PCR plate. Quantitative PCR was performed with a StepOnePlus™ real-time PCR system (Thermo Fisher Scientific). The cycling conditions were: 95°C (20 s), followed by 50 cycles of 95°C (1 s) and 60°C (20 s).−ΔΔCT method was used to analyze the fold increase.For uPA real-time PCR, 7.5 µL SYBR green mix, 0.6 µL forward primer (10 µM), 0.6 µL reverse primer (10 µM) (both primers purchased from Thermo Fisher Scientific), 4.8 µL injection water, and 1.5 µL cDNA (diluted 1:20 v/v) were added, totaling 15 µL per well of a 96-well PCR plate. The cycling conditions were: 95°C (10 min), followed by 45 cycles of 95°C (15 s) and 60°C (1 min). Finally, the melting curve was 95°C (15 s), 60°C (1 min), and 95°C (15 s). In this case, 18S was used as a reference gene  Table 1Table 1. 4/well) onto 96-well plates in RPMI medium supplemented with 10% FBS. When necessary, 100 µg/mL cetuximab was added to the wells after seeding and incubated for 30 min, 37°C, 5% CO2. Then, EGF was added to the wells of interest at 10, 50, or 100 ng/mL and the plates were incubated for 18 h at 37°C, 5% CO2. The wells were washed twice with FBS/phenol red-free medium and uPA inhibitor  was added at a final concentration of 50 µM for approximately 15 min at room temperature. After BC-11 incubation, lys-plasminogen was added at 0.5 µM, and 250 µM of plasmin-chromogenic substrate S2251  was immediately pipetted to the wells. Plates were analyzed on a Spectramax 190 plate reader  for 5 h, at 37°C, during which 405 nm reads were performed at 4 min intervals. All experiments were performed in duplicate.CASKI or C-33A cells were seeded , for 30 min, on ice. The cells were then washed with PBS and resuspended on 400 µL of FACs buffer (PBS + 5% FBS). Basal TM expression of C-33A and CASKI cells was evaluated on a FACSVerseTM machine . TM-protein expression was also evaluated after stimuli with 50 ng/mL EGF +/- cetuximab, for 6 h. The experiment was performed on an AccuriTM (BD Biosciences) flow cytometer, evaluating the percentage of positive events and mean fluorescence intensity (MFI).Flow cytometry assays were performed using an anti-TM antibody conjugated with the APC  fluorophore, which has peak excitation at 645 nm and emission at 660 nm. Briefly, 5×105/well) were seeded onto 6-well plates. After 24 h, cells were starved in FBS-free media for 10 h and treated with or without 0.5 µM glu-plasminogen, 100 µg/mL cetuximab, and/or 50 µM BC-11 for approximately 16 h at 37°C, 5% CO2. Plates were photographed with a Qimaging camera , at 10× magnification. Immediately after the photographs, cells were harvested for the Boyden chamber migration assay, which was performed using 8-μm pore polycarbonate membranes . The lower compartments were filled with RPMI media containing 5% FBS and 3×104 treated cells were seeded in 50 μL serum-free media to the upper chambers. After 20 h of incubation, at 37°C with 5% CO2, the non-migrated cells in the upper chambers were removed and the membranes were fixed and stained using the fast Panoptic staining kit . Ten image fields were evaluated for each condition. The mean number of cells per field was calculated for each condition (of each experiment) and plotted.CASKI cells ; D: larger tumor mass diameter (mm); d: smaller tumor diameter (mm).For The measurements were made using a caliper. Day 0 was defined as the day when the cell injections were made, and then tumors were measured on days 7, 9, 12, 14, and 15. Injected animals were separated into two groups: 1) Control group: treated intraperitoneally with 200 µL PBS ; and 2) Intervention group: treated intraperitoneally with 200 µL cetuximab . Both tumors in each animal were analyzed as an independent experimental unit. Treatments were performed on the same day of size measurements, except for day 15. On day 15, the animals were anesthetized with ketamine/xylazine and euthanized by cervical displacement. Immediately after euthanasia, tumors were excised using scissors and scalpel and weighed on a precision scale. Finally, tumors were cut into smaller pieces with a scissor, inserted into 1.5 mL Eppendorf tubes containing Trizol, and stored at -20°C. Samples were then macerated using a Turrax homogenizer , which was washed with DEPC water, Trizol, 75% ethanol, and DEPC water, in this order, between samples. Samples were then further processed for real-time PCR. The use of animals in this work was approved by the Commission of Ethics in Animal Use of the Federal University of Rio de Janeiro, registered at the National Control Council of Animal Experimentation under the process number 01200.001568/2013-87, protocol 102/16.t-test  or two-sample unpaired two-tailed t-test was employed. For multiple comparisons, one-way ANOVA with Tukey's post-test was used. All statistical analyses were done with Graphpad PrismTM. When applicable, data are reported as means±SD  and MEK1/2-ERK1/2 (PD98059) . EGF-indTo assess the possible functional impact that EGFR-dependent plasmin generation may have on CASKI cells, we evaluated the cell morphology under different treatments . Upon plCell migration experiments were performed immediately after cell-morphology images were taken and, although plasminogen addition led to a trend of increased cell migration on a Boyden chamber model, EGFR or uPA inhibition alone did not decrease plasminogen-induced cell migration, whereas the combination of both inhibitors significantly hampered this process .in vivo tumor growth and weight  new therapeutic targets and 2) biomarkers for screening patients that could benefit more from certain targeted therapies. EGFR has been considered to be a receptor involved in cervical cancer progression ,5, howevvs normal mucosa did not correlate with alterations in patient survival (In the present work, EGFR, uPA, and TM expression had a positive correlation on human cervical tumor samples, and the high expression of these genes was associated with worse patient overall survival. A meta-analysis published in 2002, with data from 8,377 breast cancer patients, showed that increased uPA levels correlated with worse disease-free and overall survival . Serum usurvival . In cervsurvival . Anothersurvival .in vivo tumor growth and angiogenesis (It has been shown that the uPA receptor, uPAR, can transactivate EGFR  and thatogenesis . A cliniogenesis . Anotherogenesis . PerhapsIn the present work, we showed that EGFR, uPA, and TM levels of human cervical tumors correlated with worse overall patient survival and that uPA and TM were positively regulated by EGFR. Furthermore, uPA expression can have a direct impact on cervical cancer cell function, specifically EGFR-dependent cell morphology modifications and EGFR-independent cell migration. To the best of our knowledge, we are the first to show this interplay between EGFR signaling and components of the fibrinolytic system in cervical cancer. Therefore, we propose uPA as a potentially novel candidate to be studied in combination with EGFR in cervical cancer, either as a therapeutic target or as a biomarker for EGFR signaling and patient prognosis.


This study sought to explore the role of metabolic disturbance in immunoregulation of gingivitis targeting T helper 17 cells (Th17)/regulatory T cell (Treg).+CD25+ Foxp3+Treg cells and the percentage of CD4+IL-17A+ Th17 cells in whole blood of subjects in both groups. Gas chromatography-mass spectrometry (GC-MS) was employed to analyze the plasma metabolites in the gingivitis patient group. Statistical analysis was applied to determine whether the plasma metabolites and related metabolic pathways significantly differed between gingivitis patients and healthy controls. Ingenuity pathway analysis (IPA) was employed to identify the potential relation between the metabolites and the Th17 and Treg related pathway.A total of 20 gingivitis patients and 19 healthy volunteers were recruited. Quantitative real time polymerase chain reaction (qRT-PCR) was used to evaluate expression patterns of Forkhead box protein P3 (Foxp3), transforming growth factor-β (TGF-β), retinoid-related orphan receptor-gammat (RORγt) and interleukin 17A (IL-17A) in the peripheral blood lymphocytes of subjects across the two groups. Moreover, the enzyme-linked immunosorbent assay (ELISA) technique was used to detect levels of TGF-β, IL-4, IL-6,TL-10 and L-17A secreted in the plasma as well as the SIgA secreted in saliva. Flow cytometry was used to detect the percentage of CD4+IL17A+Th17 cells and IL-17 significantly increased in the peripheral blood in the gingivitis group. Moreover, the upregulation of IL-17A mRNA and RORγt mRNA were also found in the gingivitis group. However, the percentage of CD4+CD25+ Foxp3+Treg cells and Foxp3 mRNA in the whole blood did not significantly change. However, TGF-β mRNA as well as TGF-β, IL-4, IL-6, IL-10 in the periperial blood and SIgA in the saliva were higher in the gingivitis group. Notably, that the ratio of Th17/Treg cells was significantly increased during peripheral circulation. Furthermore, we identified 18 different metabolites which were differentially expressed in plasma between the gingivitis and healthy control groups. Notably, the levels of cholesterol, glycerol 1-octadecanoate, d-glucose, uric acid, cyclohexaneacetic acid, 3-pyridine, tryptophan, and undecane 2,4-dimethyl were significantly up-regulated. whereas the levels of lactic acid, glycine, linoleic acid, monopalmitic acid, glycerol, palmitic acid, pyruvate, 1-(3-methylbutyl)-2,3,4,6-tetramethylbenzene, 1 5-anhydro d-altrol, and boric acid were down-regulated in the gingivitis group, relative to healthy controls. IPA showed that these metabolites are connected to IL17 signaling, TGF-B signaling, and IL10 signaling, which are related closely to Th17 and Treg pathway.The percentages of CD4Overall, these results showed that disturbance to glycolysis as well as amino and fatty acid metabolism are associated with Th17/Treg balance in gingivitis. Impaired immunometabolism may influence some periodontally involved systemic diseases, hence it is a promising strategy in targeted development of treatment therapies. Epidemiological studies have estimated that more than 82% of juveniles in the United States exhibit gingivitis and gingival bleeding . In ChinPeriodontitis is a chronic infectious disease that starts as an inflammation of the periodontal tissues, eventually causing resorption of alveolar bone and subsequent loss of teeth. The disease is characterized by inflammation and bleeding of the gums, periodontal pocket formation, and gradual loss of attachment. Dental plaque biofilm is a major etiological factor associated with the development of periodontitis. Generally, periodontal pathogens enter periodontal tissues and intentionally or unintentionally cause differentiation of osteoclasts as well as absorption of alveolar bone, thereby facilitating development of periodontitis, and activating intrinsic cells. Consequently, the activated T and B cells initiate adaptive immunity by antigen-presenting cells after 21 days of innate immunity. Previous studies have demonstrated that conversion of gum tissue lesions into periodontitis is mediated by T cells .Interestingly, recent studies have shown that gingivitis and periodontitis are correlated with imbalance of Th17/Treg-related cytokines . NotablyTo date, immune cells are considered highly dynamic with regards to their growth, proliferation, and effector functions during response to immunological challenges. In fact, different immune cells may adopt different metabolic configurations that enable the cell to balance its energy, molecular biosynthesis, and longevity requirements . PreviouThis observational study enrolled 20 gingivitis patients and 19 healthy volunteers at the Zhejiang Chinese Medical University and the Second Affiliated Hospital of Zhejiang Chinese Medical University between December 2016 and December 2017. The study protocol was approved by the Ethics Committees of Zhejiang Chinese Medical University (No.2014zjtcm-001). All participants provided informed consent prior to recruitment in the study.Participants were eligible if they were aged between 18 and 60 years, had at least 20 teeth with a clinical sign of generalized plaque-induced gingivitis, and signed a written informed consent form.Participants were excluded from the study if they: (a) were or planned to get, or were at risk of becoming pregnant without contraception; (b) were diagnosed with tumors, severe disease, immune, mental, and other diseases of the target organs of each system; (c) had undergone orthodontic surgery or tooth extraction three months prior to start of the study; (d) were taking antibiotics; and (d) had a history of drug abuse.Five ml of venous blood was drawn from subjects, on an empty stomach, between 6:00 -8:00 in the morning. 1 ml of the blood was collected in a tube with heparin sodium anticoagulant and used for flow detection, while the remaining 4ml was mixed with ethylene diamine tetra-acetic acid (EDTA) and used to separate plasma and peripheral blood mononuclear cells (PBMC) by Lymphocyte Separation Medium  according to the manufacturer’s instructions. In brief, 4 ml of lymphocyte separation solution was added into an appropriate centrifuge tube, under aseptic conditions, mixed with an equal amount of PBS, and the mixture carefully added above the level of the lymphocyte separation fluid. The contents were centrifuged at 1500 rpm, for 15 minutes at room temperature. The centrifuge tube was taken out, and the mixture divided into four layers from top to bottom, with the first, second, third and fourth layers representing plasma, PBMC, transparent separation liquid layer, and red blood cells, respectively. The plasma and PBMC were divided into several microfuge tubes (1.5 ml each) and stored at -80°C until further experiments. We also recorded the name, gender, age, weight, and severity of gingivitisParticipants were asked to gently rinse their mouth after half an hour, and 2 ml of saliva collected into sterile containers. The saliva was aliquoted into 1.5 ml microfuge tubes and stored at - 80°C within one hour of collection for later use.-△△CT method.Total RNA was isolated from PBMCs using the TRIzol reagent , according to the manufacturer’s instructions, and its concentration determined using a NanoDrop spectrophotometer . 500 ng of the total RNA was reverse-transcribed into complementary DNA (cDNA) using the PrimeScript RT Master Mix kit , then used for quantitative real time polymerase chain reaction (qRT-PCR) analysis using the LightCycler 480 SYBR Green kit . Amplification was done targeting the TGF-β, Foxp3, RORyt and IL-17A genes. Primer sequences for these genes are outlined in Levels of TGF-β and IL-17A in plasma as well as SIgA in saliva were determined as described in manufacturers’ protocols of the Human TGF-β ELISA Assay kit , Human IL-17A ELISA Assay kit  and Human SIgA ELISA Assay kit , respectively, then compared between gingivitis patients and healthy controls Optical density (OD) for each sample was determined at 450 nm wavelength within 15 min using a multifunctional enzyme plate analyzer. We used the standard concentration as the abscissa and the corresponding OD value as the ordinate to draw a standard linear regression curve, then calculated concentrations value of each sample according to the curve equation.4 events were acquired for each sample via flow cytometry.Fresh heparinized peripheral blood samples were collected from each group. 200 µl of whole blood sample was collected in a flow tube, based on pre-test and reagent instructions, and subjected to cell staining. The contents were incubated with 10 µl anti-human CD4  and 10 µl anti-human CD25  for 30 min at room temperature.Later, 2 ml 1×FCM Lysing Solution  was added to each tube and incubated at room temperature for 15 min in the dark. The product was centrifuged at 350g for 5 min, and the supernatant was discarded. Then 2 ml PBS was used for washing 3 times. Next,1 microliter 1× Intracellular Fixation & Permeabilization Buffer  was added at room temperature in the dark for 60 min. Thereafter, the contents were mixed with 1 ml 1×Permeabilization Buffer , centrifuged at 350 g at 4°C for 6 minutes, the cells resuspend in 100 µl 1×Permeabilization Buffer. 15 µl of anti-human Foxp3  and rat IgG2a K Isotype Control  were added to the tube, followed by a 30-min incubation at 37°C in the dark. An additional 2 ml 1×Permeabilization Buffer was added to the contents, followed by centrifugation for 6 min at 350 g at 4°C. Finally, the cells were resuspended in 500 µl Flow Cytometry Staining Buffer  and subjected to flow cytometry on the flow cytometer . A total of 5×102. Thereafter, 1.5 μl of Leukocyte Activation Cocktail with BD GolgiPlug™  was added to each well, and incubated for 4.5 h. The cultures were incubated with anti-human CD4  for 15 min at room temperature in the dark. And then,100µl MEDIUM A of FIX&PERM Kit was added and incubated at room temperature for 15 min. Three ml Flow cytometry Staining buffer was used for washing. Later, 100µl MEDIUMB of FIX&PERM Kit was added and mixed. After fixation and rupture, cells were incubated with anti-human IL-17A  for 15 min at room temperature in the dark. Three ml Flow cytometry Staining buffer was used for washing. Finally, the cells were resuspended in 500 µl Flow Cytometry Staining Buffer and subjected to flow cytometry on the flow cytometer. Non-stimulation of the Leukocyte Activation Cocktail of the Th17 cells from one healthy control subject was used for biological control. Data were analyzed using the FlowJo software 10.0 .Th17 cells were also detected by flow cytometry. Briefly, 250 μl heparin anticoagulated whole blood was inoculated into RPMI-1640 cell culture media, supplemented with 10% fetal bovine serum and 1% double-antibody, and cultured in a 24-well plate at 37°C in a 5% COPlasma samples were first thawed and homogenized at room temperature, Then 200 μL cold methanol with an internal norm applied to 50 μL of plasma for protein precipitation, in an ice-water bath. The contents were mixed by vortexing, for 30 s, centrifuged at 12,000 g at 4°C for 10 min, the supernatant transferred to microfuge tubes and freeze-dried using the Labconco CentriVap system  and incubated for 90 min in a water bath at 40°C. Thereafter, 40 μL of MSTFA was added and incubated for 60 min for trimethylsilylation. To verify the repeatability and stability of the sample analysis, for every 10 real samples, quality control (QC) samples were added to the analysis chain. The QC sample was prepared by combining the plasma samples tested similarly, with these samples collected and analyzed alongside the real samples.GC–MS analysis was performed on an Agilent 7890/5975C GC–MS , with chromatographic separation achieved using a DB-5 fused silica capillary column (30 m × 0.25 mm × 0.25 μm) . The flow rate of the carrier gas  was 1.2 mL/min, and the column oven heating was programmed as follows: after initial holding at 70°C for 3 min, the temperature was increased to 300°C at a rate of 5°C/min, and kept it maintained there for 5 min. The injection volume and split ratios were 1 μL and 5:1, respectively, while the injection port and transfer line temperatures were 300 and 280°C, respectively. The ion source (EI) temperature was 230°C, whereas the mass spectrum scan range and solvent delay time were m/z 33~600 and 4.8 min, respectively. All samples were analyzed in a random order, and QC samples were injected within the sequence.P-value of the t-test (P < 0.05). Qualitative differential analysis of metabolites was performed using the following method: search an online database (Metlin) (compare mass-to-charge ratio m/z or accurate molecular mass) and input the total difference substances obtained into MetaboAnalyst 3.0 to obtain the metabolic pathways where these different substances are located.Raw data from subjects in both groups were analyzed using the Mass Profiler Professional (Agilent) software. The data were normalized and post-edited using Microsoft EXCEL 2010 software, and final results organized into a two-dimensional data matrix, including variables , molecular weight (mass), observations (sample), and peak intensity. The edited data matrices were imported into SIMCA-P software (version 13.0) and subjected to unsupervised principal component analysis (PCA) as well as supervised partial least squares discriminant analysis (PLS-DA) for pattern recognition. We adopted the supervised method orthogonal partial least-squares discriminant analysis (OPLS-DA) for modeling to distinguish between the gingivitis and control groups. To identify differentially expressed metabolites, we applied the variable importance in projection (VIP) value  of the OPLS-DA model, coupled with the http://www.nist.gov/srd/mslist.htm) to match the molecular formula and structure of the identified compounds. Finally, we performed metabolic pathway analysis of the potential biomarkers using the KEGG (http://www.kegg.com/) and HMDB (http://www.hmdb.ca/) databases.To identify significantly changed metabolites, we searched the National Institute of Standards and Technology mass spectral library  for similar metabolites. Subsequently, we used the NIST Mass Spectral Search Program Version 2.0 , while non-normal data were presented as medians with their interquartile ranges. For uniform variances, an independent-sample P < 0.001). The patients’ gingival was swollen and sore  were within the normal range. Gingivitis patients had significantly higher Sulcus bleeding index (SBI) and gingival index (GI) than their healthy counterparts , relative to the healthy control (19.74 ± 2.88 μg/ml) group Figure 52X = 0.666, Q2 = 0.427)  , to search for potential metabolites potentially causing the differences between groups, and identified 18 significantly different metabolites between the gingivitis and healthy control groups. Eight of these metabolites, namely cholesterol, glycerol 1-octadecanoate, d-glucose, uric acid, cyclohexaneacetic acid, 3-pyridinol, tryptophan, and 2,4-dimethyldecane increased from high to low in the gingivitis compared with the control group. Similarly, cluster relationship revealed that the remaining 10 metabolites, namely, lactic acid, glycine, linoleic acid, 1-palmitoylglycerol, hexadecanoic acid, arachidonic acid, pyruvic acid, 1-(3-Methylbutyl)-2,3,4,6-tetramethylbenzene, 1,5-anhydro-d-altritol, and boric acid had higher levels in gingivitis patients that healthy controls . Therefore, the increase of TGF-β along with the higher levels of RORγt, accompanied by IL-6 in peripheral blood lymphocytes, differentiate into Th17 cells. Moreover, another possible cause may be the transformation of Treg into Th17 cells.Results of the present study revealed significant upregulation of TGF-β and IL-17A in gingivitis patients, relative to healthy controls. Similarly, Foxp3 and RORγt also exhibited an increasing growing trend in the gingivitis group. ELISA results revealed higher levels of IL-17A, IL-10, IL-6 and TGF-β in plasma of gingivitis patients relative to normal controls. Due to the increase of TGF-β and FoxP3, the Treg cells, in general, should increase. However, we found CD4nd Foxp3 .In additnd Foxp3 . In our +CD25+Foxp3+Treg cells. However, we found CD4+CD25+Foxp3+Treg cells no significant difference compared with the healthy control group, possibly due to transformation of Treg into Th17 cells. The high TGF-β levels, accompanied by IL-6 in peripheral blood lymphocytes, activated Th17 cells and caused elevated percentage of CD4+ IL-17A+ Th17 cells in the peripheral blood of gingivitis patients, thereby secreting corresponding inflammatory factor IL-17A. This caused local inflammation to occur in gingivitis patients which consequently stimulated local immunity. Analysis of Th17/Treg cell percentage ratio revealed significantly higher cell numbers in gingivitis patients than in normal controls, further suggesting that Th17/Treg imbalance may be the underlying mechanism of gingivitis.Taken together, a possible mechanism of gingivitis is that an increase in TGF-β content, coupled with IL-10 upregulation, in peripheral blood lymphocytes activated Treg cells, while transcription factor Foxp3 participated in a synergistic effect, thereby producing CD4Secretory immunoglobulin A (SIgA) is a major human mucosal immune antibody, that is predominantly present in external secretions, such as saliva, tears, milk, gastric, intestinal, respiratory, and urogenital tract fluids. SIgA, with a molecular weight of about 400 kD, is composed of double IgA, J chain and secretion piece (sc) covalently. J chain and IgA (d) are produced by locally activated B cells, sc is synthesized by mucosal epithelial cells, awhile sc covalently binds with J chain to form a dimer structure with IgA (d), namely SIgA , 25. TheRecent studies have shown that SIgA is significantly elevated in mixed saliva of gingivitis patients relative to healthy controls . Other eResults of the present study revealed significantly higher levels of SIgA in saliva as well as TGF-β, IL-4 and IL-10 in plasma of gingivitis patients, relative to healthy controls, suggesting enhanced local humoral immunity. The possible mechanism for this phenomenon could be due to stimulated inflammation which mediated secretion of SIgA in the oral. Later, SIgA worked with lysozyme and complement in saliva to dissolve local bacteria and toxins in the gums. In addition, part of SIgA may combine with T cells in the body, and as a result, cause secretion of cytokines, such as TGF-β, IL-4 and IL-10 to strengthen local immune function and further mobilize the body’s own immune response to gingivitis.Previous studies have shown that periodontitis patients exhibit different metabolites in their saliva, serum, and gingival crevicular fluid, which may help unravel the underlying molecular mechanism of periodontal disease , 33. In Moreover, we observed abnormal fatty acid metabolism in gingivitis patients, as evidenced by down-regulated unsaturated fatty acids, linoleic acid, and arachidonic acid in plasma. Linoleic acid (LA) can be converted to the metabolically active arachidonic acid (AA), which plays a critical role in inducing inflammation and adipogenesis as well as endocannabinoid system regulation AA, generated from LA or dietary. In addition, arachidonic acid intake can be converted into numerous inflammatory metabolites by cytochrome P450, cyclooxygenase, and lipoxygenase pathways . Diabete+T cells (such as Th17 cells) and CD8+CTL primarily obtain energy through aerobic glycolysis and glutamine cleavage pathways. At the same time, these rapidly dividing cells also synthesize a large number of esters. However, Treg and memory CD8+T cells are similar to the initial T cells, in that they don’t depend on glycolysis but mainly rely on ester oxidation for energy provision. In the present study, it is possible that Th17 cells increased by obtaining energy from glycolysis.Glucose was significantly upregulated in the plasma of gingivitis patients, whereas pyruvate was down-regulated. Pyruvate is a central intermediate of sugar metabolism in the body, that helps determine mutual transformation of various substances in the body. Down-regulation of pyruvate in patients with gingivitis may be ascribed to the increased consumption of pyruvate, and this causes accumulation of glucose in the body, indicating abnormal metabolism of glycolysis. On the other hand, lactic acid may be produced through the gluconeogenesis pathway to produce glucose and glycogen, thereby down-regulating lactic acid in gingivitis patients. Activated CD4Through the IPA analysis, we found that these different metabolites especially D-glucose, Glycine and Pyruvic acid were closely related IL-17 signaling, TGF-B signaling, IL-6 signaling pathway, and IL-10 signaling pathway which play significant role in Th17/Treg.The underlying mechanism of local inflammation and systemic injury in gingivitis is often complex, with Th17/Treg playing an important role in progression of inflammation. The present study uncovered the role played by Th17/Treg imbalance and immune metabolism in development of gingivitis. Taken together, our findings indicate that impaired glycolysis, as well as fatty and amino acid metabolism in the plasma of gingivitis patients interact with Th17/Treg imbalance in the body  project (NO. 2014CB543001); National Natural Science Foundation of China ; Natural Science Foundation of Zhejiang Province  and Zhejiang Medicine and Health Science and Technology Project (NO. 2021KY843).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The viability and proliferation of IMR-90 cells were examined by CCK-8 and EdU incorporation assays. The differentiation of IMR-90 cells was assessed by detecting the mRNA and protein expression levels of collagen (COL)-1 and alpha-smooth muscle actin (α-SMA). Besides, immunofluorescence assay was conducted to evaluate the localization of α-SMA in TGF-β1-induced IMR-90 cells. Moreover, the combination of PLB with/without TβRI , PI3K/Akt (Ly294002) or mTOR (rapamycin) inhibitor was pretreated on IMR-90 cells after TGF-β1 induction. For in vivo studies, a rat model of TS was established. The pathological features and severity of TS were determined by hematoxylin and eosin staining. The protein levels of TGF-β1/Smad and Akt/mTOR pathways were detected for both in vitro and in vivo models. PLB effectively inhibited the proliferation and differentiation of TGF-β1-induced IMR-90 cells, and suppressed TGF-β1/Smad and Akt/mTOR signaling pathways both in vivo and in vitro. Furthermore, PLB reduced the degree of TS in rats. Taken together, our results indicate that PLB regulates lung fibroblast activity and attenuates TS in rats by inhibiting TGF-β1/Smad and Akt/mTOR signaling pathways. In conclusion, this study implies that PLB may serve as a promising therapeutic compound for TS.Traumatic tracheal stenosis (TS) is a serious respiratory disease characterized by hyperplasia of airway granulation. Plumbagin (PLB) is a natural naphthoquinone component with anti-fibrotic properties. This research aimed to explore the roles of PLB in alleviating TS and the underlying mechanisms. For    The risAfter airway injury, tissue repair can lead to the production of fibroblastic cells and collagen-based extracellular matrix proteins, and the ensuing tissue granulation represents a key factor for the development of TS. Microscopic evaluation of the airway stenosis tissue demonstrated the existence of extracellular components such as collagen, fibronectin and proteoglycans. When the fibroblast were stimulated persistently and differentiated into myofibroblast, the symbolic protein α-SMA  could help to promote the strengthening of hyperplastic granulation and once the excessive fibrotic reactions manifested as a ‘bad scar’ .TGF-β belongs to a superfamily of secreted polypeptide growth factors involved in cell proliferation, differentiation, invasion, migration and apoptosis ,11. As oPlumbago zeylanica L, Juglans regia, J. cinerea, and J. nigra. PLB has a variety of biological activities, such as apoptotic induction, anti-inflammatory, anti-fibrotic, anti-tumor and antioxidant effects  x 100%, where As, Ac and Ab are the OD of the experimental group, control group and zero-adjustment group, respectively.2.3.The effect of PLB on TGF-β1-activated IMR-90 cell growth was determined by EdU (5-ethynyl2ʹ-deoxyuridine) incorporation assay. First, IMR-90 cell line was grown into six-well plates, induced with TGF-β1 (10 ng/mL) for 2 days, and then exposed to 10 μM PLB for 1 day. After rinsing in PBS, the cell line was incubated with 1 μM EdU at 37°C for 2 h. Following incubation, the cell line was fixed with 4% paraformaldehyde for 15 min, incubated with 0.5% Triton X-100 for 15 min, and washed 3 times with PBS. Subsequently, the cell line was incubated with a 0.5 mL click additive solution at ambient temperature for 30 min in the dark. Thereafter, the cell line was stained with 1 μg/mL Hoechst 33,342 for 10 min. Lastly, all images were recorded using an Olympus fluorescence microscope .The ability of LY294002, SB-431,542 and rapamycin to further inhibit TGF-β1-induced IMR-90 cell proliferation was determined using an analogous EdU Alexa Fluor® 594 proliferation assay  in compliance with the manufacturer’s protocol. Briefly, IMR-90 cell line was grown in six-well plates, and then pretreated with 10 μM LY294002, 10 μM SB-431,542 or 100 nM rapamycin for 2 h, induced with TGF-β1 (10 ng/mL) for 2 days, and then exposed to 10 μM PLB (in 10% FBS-supplemented DMEM) for 1 day. Next, the IMR-90 cells were marked, fixed, cleaned and permeated with EdU, followed by the addition of click additive solutions and nuclear staining .2.4.ad libitum.Sprague–Dawley (SD) rats , specific pathogen free, were procured from Laboratory Animal Center, Guangxi Medical University, Nanning, China. These rats were kept under the control conditions , and then subjected to 1-week period of acclimatization before experimental testing. Food and distilled water were provided All rats were randomly classified into four groups (n = 6 per group): (1) No template control (NC) group, (2) TS (model) group, (3) TS + normal saline (TS+NS) group, and (4) TS + PLB (TS+PLB) group. The TS model was constructed based on the methods described by Mizokami and coworkers . TracheoAfter 9 days, the rats were euthanized by intraperitoneally injecting 150 mg/kg  pentobarbital. The specimens were extracted. The narrowing tracheal segment of about 1 cm was cut off for future experiments . Ethical2.5.Western blotting was conducted using standard procedures. Total protein was isolated from the treated fibroblasts and tracheal tissues using radioimmuneprecipitation assay buffer (high)-phenylmethylsulfonyl fluoride . Subsequently, total protein content was determined by BCA assay kit . An equal number of proteins were electrophoresed through SDS-PAGE, and then transferred onto nitrocellulose membranes . After inhibiting with 5% skimmed milk for 2 h, the membranes were incubated at 4°C for 12 h with the following primary antibodies: α-SMA , COL1 , Akt , p-Akt (ab19623), mTOR (ab32028), p-mTOR (ab109268), Smad2 , p-Smad2 (ab188334), Smad3 (ab40854), p-Smad3 (ab52903), TGF-β1 (ab179695) and β-actin . All antibodies were purchased from Abcam (USA), with a dilution of 1:1000, unless otherwise stated. After rinsing 3 times in TBST, the membranes were incubated with the respective secondary antibodies at ambient temperature for 2 h. Lastly, the immunoblots were detected by electrochemiluminescence plus reagent kit , and densitometric analysis of each target protein band was conducted using ImageJ software (NIH).2.6.The trachea and its surrounding soft tissues were examined macroscopically for TS assessment. The pathological alterations of the airway mucosa and the degree of the lumen stenosis were evaluated by hematoxylin and eosin (H&E) staining. The tracheal tissues were cut at 4-mm thickness and fixed with 10% formalin for over 1 day. After dehydration and clearing, the excised tissues were embedded in paraffin, followed by HE staining. The HE staining was performed as described previously . ImageJ 2.7.The mRNA levels of COL1 and α-SMA in IMR-90 cells were analyzed in biological triplicates. Total RNA was prepared using Trizol regent , and then reverse-transcribed with PrimeScript RT Master Mix . Real-time quantitative PCR was conducted on a second-generation PCR instrument  using TB Green Premix Ex Taq II (Tli RNaseH Plus). The reaction conditions were as follows: 95°C for 30 s, followed by 40 cycles of 95°C for 5 s, 60°C for 30 s and 72°C for 15 s. ACTB was employed as a reference gene for normalizing the gene expression data. 2.8.IMR-90 cell line was grown in 24-well plates with glass bottom for 1 day. Once reaching 60–70% confluency, the cells were induced by TGF-β1 (10 ng/mL) for 2 days, except NTC group, and then exposed PLB  for 1 day. After washing 3 times with cold PBS, the cells were fixed with 4% paraformaldehyde at ambient temperature for 15 min, permeabilized with 0.5% Triton-X 100 in PBS for 10 min, and then blocked with 5% bovine serum albumin in PBS. Subsequently, the cell line was incubated with anti-α-SMA antibody (1:100) at 4°C for overnight, and then incubated with the corresponding fluorescent substance-labeled secondary antibody for 1 h. After mounting with DAPI  stain, the immunofluorescene images were recorded using an inverted fluorescence microscope with phase contrast .The paraffin sections were de-waxed and hydrated in the xylene and ethanol, then washed with PBS for 3 times. Citric acid repair solution (0.01 M) was used for antigen repair in boiling water bath for 10 min. After washing three times with PBS, the tissue sections were blocked with 5% BSA at room temperature for 20 min. Next, they were incubated with COL1 antibody (1: 400) overnight at 4°C, then incubated with fluorescent antibody Cy3 (1: 200) at 37°C for 30 min. DAPI was used for counterstaining of the nucleus. After washing with PBS again anti-fluorescence quenching agent, sections were mounted avoiding exposure of the slides to light. Inverted fluorescence microscope and ImageJ software was used for recording and quantitative analysis.2.9.in vitro assays were repeated for 3 times. Statistical difference between groups was compared with one-way ANOVA followed by Newman–Keuls test for normally distributed continuous variables; or otherwise, Kruskal–Wallis test was applied. P < 0.05 was deemed to be statistically significant.SPSS V.22.0  was employed for the statistical tests. All results were presented as mean ± standard deviation (SD), and the 3.The purpose of this study was to explore the mechanism of tracheal stenosis and the effect of plumbagin on it. We hypothesized that plumbagin attenuated rat tracheal stenosis after injury by regulating the activation of fibroblast through TGF-β1/Smad and Akt/mTOR signaling pathway. We verified our hypothesis by using the rat model of tracheal stenosis caused by nylon brush injury and fibroblast experiment.3.1.To determine the effect of PLB on tracheal stenosis, we established a rat TS model. H&E staining revealed that hyperplasia of the airway epithelium and thickening of the submucosal layer could induce lumen stenosis. Infiltration of inflammatory cells, increased fibroblasts and fibrocytes in the submucosal layer were also observed. Furthermore, excessive collagen deposition, mucous gland structure and angiogenesis were existed in the thickened submucosa. The thickness of tracheal granulation was markedly higher in TS model rats than in NC rats, implying the successful construction of TS rat model. In addition, the calculated mean percentage of stenosis was remarkably lower in TS+PLB group than in TS+NS group ; P < 0.03.2.in vivo.TGF-β1 plays key roles in regulating fibrosis and scarring, mainly through its downstream Smad pathways. Smad-2 and −3 are two important regulators that contribute to tissue fibrosis via TGF-β1 activation . TGF-β1 3.3.The toxicity of PLB on IMR-90 cells was tested at different concentrations over a time frame of 1 day via cell viability assay . Given t3.4.The protein  and mRNAin vitro, the levels of Smad2, Smad3, Akt and mTOR proteins were detected in IMR-90 cells after TGF-β1 induction. Noticeably, the protein levels of p-Smad2, p-Smad3, p-Akt and p-mTOR were upregulated in TGF-β1-induced IMR-90 cells  . By phosin vitro . In the in vitro , and recin vitro ,35. mTORin vitro . Severalin vitro . In pulmin vitro  used rapTo further decipher the mechanism by which PLB ameliorates wound healing via TGF-β1/Smad and Akt/mTOR signaling, we selected TβRI inhibitor (SB431542), PI3K/Akt inhibitor (LY294002) and mTOR inhibitor (rapamycin) to pretreat in TGF-β1-induced IMR-90 cells. It was observed that the protein level of p-Akt was further decreased by PLB plus SB431542 or LY294002 , that of5.In summary, our results suggest that TGF-β1/Smad and Akt/mTOR pathways are activated during the occurrence of traumatic-induced TS. Interestingly, PLB can regulate TGF-β1/Smad and Akt/mTOR pathways to inhibit the activity of TGF-β1-induced IMR-90 cells, which also reflect a mechanism by which PLB attenuates TS in rats. This study indicates that PLB may serve as a novel therapeutic drug to reduce the degree of TS.


Tlswo, in Talaromyces leycettanus JCM12802. Tlswo was successfully expressed in both Trichoderma reesei and Pichia pastoris. Assay results indicate that TlSWO is capable of releasing reducing sugars from lichenan, barley β-glucan, carboxymethyl cellulose sodium (CMC-Na) and laminarin. The specific activity of TlSWO toward lichenan, barley β-glucan, carboxymethyl cellulose sodium (CMC-Na) and laminarin is 9.0 ± 0.100, 8.9 ± 0.100, 2.3 ± 0.002 and 0.79 ± 0.002 U/mg, respectively. Additionally, TlSWO had disruptive activity on Avicel and a synergistic effect with cellobiohydrolases, increasing the activity on pretreated corn stover by up to 72.2%. The functional diversity of TlSWO broadens its applicability in experimental settings, and indicating that it may be a promising candidate for future industrial applications.Swollenins exist within some fungal species and are candidate accessory proteins for the biodegradation of cellulosic substrates. Here, we describe the identification of a swollenin gene,  Lignocellulosic biomass or plant dry matter, has been considered an alternative to fossil fuels. However, the growth and processing of biomass feedstocks for conversion into fuels, or for other chemical production purposes, remain challenging . A succeTrichoderma reesei (1), has recently been released to the public . Additionally, we showed that TlSWO can reduce Avicel particle size and surface structure, and significantly increase synergistic activity by up to 72.2% on pretreated corn stover (PCS).Previously, we identified the thermophilic e source . In thisTalaromyces leycettanus JCM12802, the donor strain, was purchased from Japan Collection of Microorganisms RIKEN BioResource Center . Escherichia coli Trans I-T1  was used for routine gene cloning. T. reesei AST1116 and P. pastoris GS115  were used as hosts for gene expression. pPIC9 (Invitrogen) and pTrEno plasmids of were used to drive Tlswo gene expression in P. pastoris and in T. reesei, respectively. The pTrEno plasmid was constructed described by TlSWO DNA and amino acid sequences were analyzed using BLASTx and BLASTp programs2, respectively (TlSWO introns and exons were predicted using the GENSCAN Web Server3 (4 (5. Sequence assembly and estimation of the molecular mass and pI of the mature peptide were achieved using the Vector NTI Suite 10.0 software (Invitrogen). Protein molecular weight and molar extinction coefficients were estimated at the ExPASy tools page6. Multiple sequence alignments were performed using the Clustal W program from MEGA software 4.0. PROSITE7 was used to analyze protein domains and functional sites 2SO4, 1.0 g/L KH2PO4, 0.5 g/L MgSO4⋅7H2O, 0.2 g/L CaCl2, 10.0 mg/L FeSO4⋅7H2O, 30.0 g/L wheat bran, 30.0 g/L soybean meal and 30.0 g/L corncob. T. leycettanus JCM12802 genomic DNA was extracted using the DNA isolation kit (Tiangen) following the manufacturer’s instructions and was used as a template for PCR amplification. Total RNA isolation and first strand cDNA synthesis were performed as previously described (Tlswo fragment corresponding to the 21–503 amino acid sequence was amplified using primers P1 (5′- GCTCGTGCTCAGAGCAGCTGTGCAGG-3′) and P2 (5′- CTAGATTACCTAGGTAAACTGCACC-3′). Amplified PCR products were cloned into pPIC9 and pTrEno vectors using Gibson Assembly  following the manufacturer’s protocol for cloning and expression. Escherichia coli Trans I-T1 (TransGen) used for routine gene cloning, was grown at 37°C overnight in Luria-Bertani medium supplemented with 50 μg/mL of ampicillin .g medium  with modescribed . PCR ampP. pastoris GS115 as the expression host, pPIC9-Tlswo recombinant plasmids were linearized with BglII (New England Biolabs) and transformed into the expression host via electroporation. Positive transformants were screened on minimal dextrose medium at 30°C for 3 or 4 days until single colonies appeared. Single colonies were placed into shaking tubes for enzyme production using protocol provided in the Pichia Expression Kit (Invitrogen). Large-scale fermentation was performed as previously described (P. pastoris GS115 transformant containing pPIC9-Tlswo was grown at 30°C in 400 mL BMGY medium in a 1 L flask with shaking at 200 rpm for 48 h. Cells were collected and resuspended in 200 mL buffered methanol-complex (BMMY) medium with 0.5% (v/v) methanol and cultured at 30°C for 72 h with shaking (200 rpm). Methanol was added into the medium every 24 h.When using escribed . The recT. reesei AST1116 as the expression host, recombinant pTrEno-Tlswo plasmids were linearized with SbfI  and used to transform T. reesei AST1116 via electroporation. Potato dextrose (PD) plates were used for spore production and PDHX plates  were used for screen potential T. reesei transformants which were grown for 2 to 3 days at 30°C. Mandels and Andreotti medium with 5% glucose (MAG) was used as the growth medium for TlSWO expression. Subsequently, complete medium lactose (CML) was used for overexpression of the transformants. MAG and CML medium protocols were previously described by ∗vol–1*min–1 was used to purge while the system was kept at a constant temperature of 28°C, and a pH of 4.8 for 48 h by using 2M KOH and HCl  sample buffer  with 50 μL/mL β-mercaptoethanol as a reducing agent. Samples were incubated at 95°C for 5 min before loading onto NuPAGE SDS gels with MOPS buffer, and proteins were electrophoresed at 200 V for approximately 40 min.Fermentation broths were harvested and sequentially vacuum-filtered. Filtered broth was then concentrated by tangential ultrafiltration with a 10 kDa MWCO . The broths were roughly concentrated to volumes of 100 mL. The final concentrated volume was exchanged with at least 2.0 L of 20 mM Bis-Tris pH 6.5 to remove residual peptides and other low molecular weight debris. The following purification steps were then performed as previously described . The cruTlSWO protein. Proteins were separated on a 12% gel and visualized by Coomassie Blue staining. Protein concentration was measured using a NanoDrop 2000 Spectrophotometer  and the Bradford protein assay kit (Bio-Rad).Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed to assess purity of the TlSWO activity was measured using the 3,5-dinitrosalicylic acid assay  in a buffer of different pH, 100 mM glycine-HCl (pH 1.0–3.0), McIlvaine buffer (pH 3.0–8.0) and glycine–NaOH (pH 9.0–12.0). For pH stability, TlSWO was preincubated at 37°C for 1 h in buffers of different pH (1.0–12.0) and subjected to the residual activity assay. The optimum temperature of TlSWO activity was determined in a reaction solution of pH 4.0 using a temperature range from 30 to 80°C. The TlSWO thermostability assay (100 μg/mL) was performed out by preincubating the enzyme at 37, 50, 60, or 70°C for 0–60 min, and performing residual activity assays on 100 μL aliquots withdrawn at different time points.The effects of pH and temperature on TlSWO in 100 mM citric acid-Na2HPO4 buffer (pH 4.0). The experiment was performed on a rotary shaker at 40°C for different time intervals. Control experiments without TlSWO were also performed under the same conditions. The physical structure of Avicel fibers was initially observed using light microscopy . Subsequently, photomicrographs of the samples were captured using a scanning electron microscope  at a voltage of 15 kV.Avicel PH-101 was used as a solid cellulosic substrate. Avicel (10 mg) was incubated with different amounts of purified Hydrolysis reactions on 1% barley β-glucan, 1% CMC-Na, 1% laminarin and 0.5% lichenan, were performed overnight at pH 4.0 and 37°C using the 100 μg/mL of the enzyme. High-performance anion-exchange chromatography  equipped with a Carbo-Pac PA200 column (3 × 250 mm) was used to determine the reaction products released from the polysaccharide.g for 10 min. The supernatant was then decanted and the pellet resuspended in deionized (DI) water until the pH reached 5.0. CNCs were pelleted by centrifugation, collected and resuspended in DI water. Each substrate was suspended in 20 mM sodium acetate buffer, pH 5.0 and reactions were performed in triplicate in vials at 40°C. Avicel PASC was prepared as described by Trichoderma longibrachiatum (Megazyme Co.), cellobiohydrolases Cel7A from Penicillium funiculosum and β-glucosidase from Aspergillus niger (Megazyme Co.) at concentrations (mg protein/g of glucan) of 2, 10, and 1, respectively. The reaction was carried out for 120 h, with sampling every 24 h. Samples of 100 μL, containing both solids and liquid were removed and diluted for HPLC sugar analysis using a Bio-Rad HPX-87H column. Control experiments using bovine serum albumin were also performed under the same conditions.The substrates used in this work were National Renewable Energy Laboratory (NREL) dilute acid PCS P120927, cellulose nanocrystals (CNCs) and phosphoric acid swollen cellulose (PASC). Each substrate was equivalent to 8.5 mg of glucan. For CNC preparations from Avicel, about 2 g of Avicel was added to HCl pre-heated at 80°C. Then, the acid hydrolysis was run for 4 h, and stirred every 15 min using a glass or Teflon rod, followed by centrifugation several times at 1,600 × Tlswo open reading frame consists of six exons and encodes a 503 amino acid protein (TlSWO) and a signal peptide at the cleavage site between amino acids 20 and 21. The cloned gene sequence encoding a putative TlSWO was submitted to NCBI GenBank as MT180127. Deduced TlSWO shared 71.5% sequence similarity to the swollenin (BAI83433.1) from A. fumigatus, 71.2% to the swollenin (ADZ74267.1) from P. oxalicum. Further analysis using PROSITE demonstrated that TlSWO consists of three domains, fungal-type carbohydrate-binding module family 1 (CBM1) (amino acids 23−59), family 45 endoglucanase-like domain of expansin (Expansin_EG45) (amino acids 206−388) and a cellulose-binding-like domain of expansin (Expansin_CBD) (amino acids 400−492), all of which are typical of swollenins from fungi . CBM1 and Expansin_EG45 are connected by a Serine-Threonine rich linker domain. Although the function of the linker has been studied in other cellulases, it is not clear if the linker plays a similar role in swollenins  catalytic motif of the GH45 cellulase , which is part of the active site  was expressed in P. pastoris and T. reesei using the aox1 and eno promoters, respectively . After Endo H digestion, the molecular weight of TlSWO decreased to ∼72 kDa, which is still higher than theoretical MW  and barley β-glucan (8.9 ± 0.100 U/mg), followed by CMC-Na (2.3 ± 0.002 U/mg). In contrast, a very low level of activity was observed with laminarin substrate (0.79 ± 0.002 U/mg)  . TogetheTlSWO activity were investigated with lichenan as a substrate. Although the enzyme displayed activity across a broad pH range (2.0–12.0), we determined that the optimal pH for TlSWO is 4.0  and scanning electron microscopy (SEM). LM analysis showed that after incubation with different amounts of TlSWO for 24 h, the Avicel’s physical structure significantly differed from that of untreated Avicel , cellobiohydrolase (EC 3.2.1.91) and endoglucanase (EC 3.2.1.4) in the presence of TlSWO. Reactions with BSA and without TlSWO were used as controls. A total of 13 mg protein/g of glucan was used in all reactions. Endoglucanases randomly cleave internal β-1,4-glycosidic bonds to create new reducing ends. This allows cellobiohydrolases to continuously act on the chain termini to release cellobiose, and β-glucosidase then hydrolyzes cellobiose into glucose . TlSWO has the highest activity on lichenan and barley β-glucan substrates, both of which contain β-1,4 and β-1,3 linkages, and its activity is minimal on laminarin, a substrate that only contains β-1,3 linkages. This indicates that the primary mechanism of action of TlSWO is via activity on β-1,4 linkages. Expansins are more highly similar to GH45 subfamily C enzymes than to other members of the GH45 family (TlSWO (HMD). The aspartic acid in this motif plays the role of proton donor in GH45. Nevertheless, other key residues that are critical for catalytic activities are absent in both expansins and swollenins. This difference suggests expansins and swollenins may use an inverted mechanism during the catalytic process. In 2015, PcCel45A, which belongs to GH45 subfamily C, uses an imidic acid form of asparagine residue as a general base in the “Newton’s cradle” proton relay catalytic mechanism. This proposal sheds light on some potential mechanistic properties of expansin’s catalytic process. Horizontal gene transfer drives sequence differences between fungal swollenins and plant/bacterial expansins  that shows the highest similarity toward fungal GH6 family 1 CBMs. Six typical conserved cysteines present in the sequence may form three pairs of disulfide bonds. The TlSWO CBM also contains three conserved aromatic residues , which are typical in GH6 and GH7 cellulase CBMs. These sequences are important for cellulase stability and activity during the reaction. Moreover, the linker length is crucial for cellulase activity . Using this model, one proposal hypothesizes that C1 can disrupt cellulose by displacing hydrogen bonds in the microfibril, leading to a more available structure for Cx  can be found below: HZ and YW performed the experiments. FZ and RB designed and performed the synergism experiments and analyzed the data. BY and XX designed the research and participated in the bioinformatics analysis. FZ and HL revised the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.