Proteomic analysis identifies post-translational functions of proteins, which remains obscure in transcriptomics. Given the important functions of Th9 cells in anti-tumor immunity, we performed proteome analysis of Th9 cells to understand the involvement of proteins that might be crucial for the anti-tumor functions of Th9 cells. Here we performed a comprehensive proteomic analysis of murine Th0 and Th9 cells, and identified proteins that are enriched in Th9 cells. Pathway analysis identified an abundance of phosphoproteins in the proteome of Th9 cells as compared to Th0 cells. Among upregulated phosphoproteins, Ppp2ca  was found to be highly enriched in Th9 cells. Although the role of PP2A has been shown to regulate the differentiation and functions of Th1, Th2, Th17 and Tregs, its role in the differentiation and functions of Th9 cells is not identified yet. Here we found that PP2A is required for the induction of Th9 cells, as PP2A inhibition leads to the suppression of IL-9 and expression of key transcription factors of Th9 cells. PP2A inhibition abrogates Th9 cell-mediated anti-tumor immune response in B16-OVA melanoma tumor model. Thus, we report that PP2A is essential for the differentiation and anti-tumor functions of Th9 cells. Th9 cells play a critical role in promoting anti-tumor immunity, allergic inflammation and autoimmune diseases4. So far, different transcription factors have been reported to play a crucial role in the development of Th9 cells11, however, these factors have also been reported in other effector T cell subsets and are not lineage specific-transcription factors for Th9 cells. Understanding the generation of Th9 cells will allow modulating effector functions of these cells during the pathophysiology of asthma and anti-tumor immune responses. Thus, identification of biomarkers for Th9 cells is crucial for developing diagnostics and therapy for promoting the clinical outcomes for patients with Th9-mediated diseases.Cytokines such as TGF-β1 and IL-4 together skew naïve CD412. Therefore, despite of several transcriptome analysis, global proteome profiling has gained significant importance for mapping protein signatures at cellular level to unravel their precise functional characterization. There have been significant advancements in the development of proteome for different immune cells. Although the mass spectrometry studies on Th cells are rare, still few studies have reported the proteome analysis of different effector T cell subsets 17.For deciphering the complete identity of a cell, quantitative proteomics is an important tool, as there is a discordance between transcriptome and proteome due to post-translational modifications and differential rates of protein synthesis. The mRNA level of a gene is poorly correlated with the protein levels in different conditions due to various above mentioned reasons15. They identified 14 differentially expressed proteins in human Th1 and Th2 cells among which N-terminal acetylation of cyclophilin A was significantly higher in Th1 as compared to Th2 cells15. Riaz et al. identified the proteome of gut-derived human Th1 and Th1/Th17 clones from Crohn’s disease patients and identified 334 differentially expressed proteins with majority of cytotoxic proteins being overrepresented in Th1 clones than in mixed phenotype Th1/Th17 clones. The Th1 clones showing cytotoxic features were CD28+ NKG2D− CD4+ Th1 cells and constituted only a subgroup when tested from seven different Crohn’s disease patients16. There are studies showing quantitative proteomic analysis of human regulatory T cells13 and during human Th17 cell polarization12. Recently published study has revealed the differentially expressed proteins in multiple sclerosis patients through quantitative proteomics of CD4+ and CD8+ T cells18. However, at present no such studies have investigated the proteomic analysis of Th9 cells.Rautajoki et al. has demonstrated the differential protein expression patterns in human Th1 and Th2 cellsHere we identified, through proteomic analysis, protein phosphatase PP2A as one of the differentially upregulated protein in murine Th9 cells. We show, here, that PP2A is essential for Th9 cell differentiation and Th9-cell mediated anti-tumor immunity.C57BL/6 (#00064) wild type (WT) and OT-II TCR (#004194) mice were procured from Jackson laboratory, housed and maintained in pathogen-free small animal facility at Translational Health Science and Technology Institute, Faridabad, India. All the mice used for experiments were 6–12 weeks old and both age and sex matched. All animal experiments were performed in accordance to the THSTI Animal Ethical guidelines and the experimental protocols were approved by institutional (THSTI ) animal ethics committee (IAEC).+CD62L+) T cells were activated with plate bound anti-CD3  and anti-CD28  in 96 well flat bottom plates and in vitro differentiated into Th0 (without cytokines) and Th9 cells in the presence of TGF-β1 , IL-4  and anti-IFN-γ  for 3 days with or without Okadaic acid  or LB-100 .6–12 weeks old WT mice were euthanized, spleen and lymph nodes (LN) were collected aseptically. Single cell suspension was prepared, then stained with the antibodies- anti-mouse CD4 PerCP , anti-mouse CD62L APC  and anti-mouse CD25 PE . Cells were sorted on BD FACS Aria with > 98% purity. Sorted purified naïve , ionomycin  and monensin  for 6 h. Cells were stained with anti-mouse CD4  and anti-mouse CD8α  antibodies followed by live/dead staining. Cells were stained with anti-mouse IL-17A , anti-mouse IL-9  or anti-mouse IFN-γ . Cells were acquired on FACSVerse (BD biosciences) and the results were analyzed with FlowJo software (Tree star).20. The excised bands were destained with 40 mM ammonium bicarbonate (ABC) in 40% acetonitrile (ACN). The gel bands were processed further for reduction using 5.0 mM dithiothreitol (DTT) (60 °C for 45 min) and alkylation using 20 mM iodoacetamide (IAA) (room temperature for 10 min in dark)20. The gel sections were dehydrated with 100% ACN followed by trypsin digestion for 10–12 h at 37 °C 20. The peptides were extracted from the gel pieces with 0.4% formic acid twice, once in 50% ACN solution and once in 100% ACN. The peptides were then vacuum-dried and stored at − 80 °C for LC–MS/MS analysis20.30 µg proteins from each of Th0 and Th9 cells were resolved by 10% SDS-PAGE followed by commassie blue staining. 21 gel pieces were excised for each of the Th0 and Th9 conditions and in-gel digestion was carried out as described previously20. Mobile phase A was 0.1% formic acid in water and 0.1% formic acid in ACN was used as mobile phase B. The elution of fractions were performed from the analytical column at a flow rate of 250 ml/min using an initial gradient elution of 10% B from 0 to 5 min, transitioned to 40% over 120 min, ramping up to 90% B for 5 min, holding 90% B for 10 min, followed by re-equilibration of 5% B at 10 min with a total run time of 150 min20.All fractions were subjected to 5,600 Triple-TOF mass spectrometer which is directly linked to reverse-phase high-pressure liquid chromatography Ekspert-nanoLC 415 system (Eksigent). The trap column (200 μm × 0.5 mm) and the analytical column (75 μm × 15 cm) were both from Eksigent, packed with 3 μm ChromXP C-18 (120 Å), and were used for reverse phase elution by Ekspert-nanoLC 415 systemm/z, followed by MS/MS scans of 20 ion candidates per cycle with activated rolling collision energy, operating the instrument in high sensitivity mode using the Analyst TF 1.7 where each 1 s MS survey scan was followed by 3 MS/MS scans of 3 s. The selection criteria for the parent ions included the intensity, where ions have to be greater than 150 cps, with a charge state between + 2 and + 5, mass tolerance of 50 mDa and on a dynamic exclusion list were present20.Peptides were injected into the mass spectrometer using 10 μm Silica Tip electrospray Pico Tip emitter (New Objective Cat. No. FS360-20-10-N-5-C7-CT), and the ion source was operated with the following parameters: ISVF = 1950; GS1 = 20; CUR = 12. The data dependent acquisition experiments was set to obtain a high resolution TOF–MS scan over a mass range 100–1,250 Mass spectra (MS) and tandem mass spectra (MS/MS) were recorded in positive-ion and high-sensitivity mode with a resolution of ~ 35,000 full-width half-maximum. Before running samples in the mass spectrometer, spectra calibration was done after acquisition of every sample using dynamic LC–MS and MS/MS acquisitions of 100 fmol β-galactosidase. Once an ion had been fragmented by MS/MS, its isotopes were excluded from further MS/MS fragmentation for 12 s. The ion accumulation time was set to 250 ms (MS) and to 70 ms (MS/MS). The collected raw files spectra were stored in .wiff format.21. MaxQuant search algorithm uses separate mass tolerances for each peptide, however, the initial maximum precursor mass tolerances were set to 20 ppm in the first search and 4.5 ppm in the main search. The fragment mass tolerance was set to 20 ppm21. Further, following parameters were used: instrument type 5,600, minimum peptide length was set as 7 and maximum peptide mass was 4,600 Da, centroid half width 35 ppm, label free quantification (LFQ) was checked with LFQ minimum ratio count 2, maximum allowed missed cleavages 1 and trypsin was specified as protease.Raw mass spectrometry files were searched in MaxQuant (version 1.6.7.0) software against a UniProt mouse protein database . Carboamidomethylation of cysteine was used as fixed modification while methionine oxidation and N-terminal acetylation were used as variable modification12. We filtered the data to keep only highly reliable stringent proteins quantitation values by using following parameters which includes: (1) threshold of 1% accepted FDR proteins; (2) ≥ 5 razor and unique peptides; and (3) absolute iBAQ > 20 intensity, was considered for analysis. Fold change (FC) was calculated by using the formula: [Th9(iBAQ)/Th0(iBAQ)]. Log10 FC values were then calculated and threshold was set as 10 FC . Proteins identified only in Th9 cells were considered highly expressed and assigned a log10 FC value of + 3 and for proteins highly expressed in Th0 cells were assigned a log10 FC value of -3.The output of this search was a .txt file which consists of protein name, accession, number of common and unique peptides, molecular weight of the proteins, sequence coverage by mass-spectrometry identification, MS/MS counts and iBAQ (intensity-based absolute quantification). The unique and razor peptides were used for the quantification of proteins. iBAQ intensities were calculated using MaxQuant software10 FC =  + 1 were subjected to GO and pathway analysis. GO analysis was done using PANTHER (https://pantherdb.org/)22 and pathway analysis was done using STRING 11 database server (https://string-db.org/)23.The top upregulated proteins at log23. The corresponding protein–protein interaction network was constructed when we selected the interactions pertaining to mouse and showed minimum interactions with a confidence score > 0.9. Differentially expressed proteins were used to create two separate networks—one for upregulated proteins while the other for downregulated proteins. The networks were manually analyzed to find out signaling related hub proteins that may be central to Th9 cell differentiation.For the identification of functional protein enrichment and associations, the network of key genes was built by using the STRING 11 database® FAST qPCR Master Mix (2×) Universal Kit (KK4600) on Fast 7500 Dx real-time PCR system (Applied Biosystems) according to manufacturer’s protocol. The relative gene expression of each target gene was calculated after normalization to the internal control (β-actin) as described previously24. The following SYBR primers were used for the analysis: β-actin , Il9 , Spi1 (forward 5′-CATGAGGTGAAATGTGAGAG-3′); reverse (5′-AGTTGGTTGAAATGGATCAC-3′), Irf4 , Hif1α , Batf , Gata3 , Irf1  and Foxo1 .RNA isolation from T cells was performed using the RNAeasy Mini Kit  and reverse transcribed into cDNA using the iScript cDNA synthesis kit . RT-PCR was done using the KAPA SYBR+ T cells were stained with 5.0 μM CFSE , and differentiated into Th9 in the presence or absence of increasing doses of LB-100  μM for 3 days. Cell proliferation was assessed by flow cytometry at the end of culture25.Naive CD4+ T cells were transfected with silencer select predesigned 25 nM siRNA specific for mouse PP2A  or silencer negative control scramble siRNA  with transfection reagent  according to the manufacturer’s instruction8 and were then differentiated into Th0 and Th9 respectively for further analysis.Naive CD45 B16-OVA cells were subcutaneously injected into flank region of WT mice for melanoma development. 2 × 106 OVA-specific OT-II-Th9 cells ± LB-100 were transferred intravenously into B16-OVA-tumor bearing mice at day 7. Mice were then randomized into following groups: Group I: mice injected with B16-OVA cells only (B16-OVA); Group II: mice injected with B16-OVA and adoptively transferred OT-II-Th9 cells (B16-OVA + Th9); and Group III: mice injected with B16-OVA and adoptively transferred OT-II-Th9 cells differentiated in the presence of LB-100 (B16-OVA + Th9 + LB-100). Tumor growth was monitored and tumor volume was measured using vernier caliper. Tumor volume was calculated as: Volume (mm3) = L × W2/2, where L is the length and W is the width of the tumor (in mm). Mice were euthanized when the tumor volume exceeded 2000 mm3 or there was severe skin necrosis defined as the end-point of the study8. At the end point, spleen and tumor draining lymph nodes and TILs were isolated26. Cells were re-stimulated ex vivo with PMA/ionomycin followed by intracellular cytokine staining in CD4+ and CD8+ T cell populations8.2 × 10P value < 0.05 was considered statistical significant for all the experiments. All the data are represented as mean ± SEM.One-way ANOVA for comparison of means between more than two groups and two-way ANOVA test for comparison among multiple groups with two variables was used with Tukey’s multiple comparison’s test for all statistical analysis using GraphPad Prism 7.0. Transcriptomics data identified essential factors that are required for differentiation and functions of Th9 cells. However, transcriptomics analysis of Th9 cells failed to capture the proteins that are modulated by post-translational modifications such as phosphorylation, ubiquitination and acetylation. To understand the proteome of Th9 cells, we performed proteome analysis, using in-gel digestion and liquid chromatography-mass spectrometry (LC–MS), of Th9 cells and compared it to the proteome of Th0 cells. This experimental design, as represented in Fig. 27, so we polarized Th9 cells in the presence of TGF-β1 and IL-4 together with the increasing concentrations of anti-IFN-γ antibody, however we did not find any difference in the IL-9 levels upon IFN-γ neutralization . This resulted in the identification of 118 upregulated proteins  in Th9 cells and found a decrease in Il9 expression by qPCR as well as reduction in the intracellular IL-9 production in Th9 cells 10, IRF411, HIF1α9, BATF7, GATA36, IRF18 and Foxo15. PP2A interactome suggests a potential direct and indirect association of PP2A with the key transcription factors of Th9 cells such as PU.1 (Spi1), IRF4, HIF1α, BATF, GATA3, IRF1 and Foxo1, all of these transcription factors are found to be prevalent in Th9 cells . Knockdown of PP2A in Th9 cells showed a significant reduction in IL-9 induction at both mRNA and protein levels as compared to control siRNA Fig. c, d. PP229. To understand whether inhibition of PP2A abrogates the anti-tumor functions of Th9 cells, naïve CD4+ T cells from OT-II TCR transgenic mice differentiated into Th9 cells in the presence or absence of LB-100. These cells were then adoptively transferred into a B16-OVA tumor bearing mice on day 7, and the tumor progression was monitored. It was found that tumor weight and volume was higher in the group of mice which received LB-100-treated OT-II Th9 cells as compared to the group of mice which received OT-II-Th9 cells alone  group as compared to OT-II-Th9 group  in the (OT-II-Th9 + LB-100) group as compared to Th9 group . In spleen, the frequency of CD8oup Fig. c. There een Fig. c. Intereoup Fig. d indicatoup Fig. e. These 30, thus we tested whether PP2A mediated inhibition of Th9 cells abrogates its anti-tumor functions via suppressing IFN-γ production. OT-II-Th9 group treated with LB-100 showed a decrease in the IFN-γ expression in CD8+ T cells in spleen, dLN and TILs as compared to OT-II-Th9 group respectively  as the most connected phosphoprotein in Th9 cells among 22 phosphoproteins and has been shown to be involved in T cell differentiation. Ppp2ca was shown to be upregulated in 25 different pathways, while Ptpn11 in 19, Plcg1 in 14 and Sec13 in 13 different pathways, among other highly expressed proteins in Th9 cells. Among top downregulated pathways manually picked, phosphoproteins showed strong protein–protein interactions with proteins which were either nucleotide-binding or involved in ubiquitination or both.32. The PP2A holocomplex takes part in the regulation of key cellular processes, such as cellular metabolism, cell cycle, apoptosis, migration, proliferation and differentiation, and is involved in the development of cancer33, SLE34 and neurodegenerative diseases35. PP2A modulates several signaling cascades, including the PI3K-AKT-mTOR 37, MAPK39, and NF-κB pathways.Protein Phosphatase-2A (PP2A) is a highly conserved heterotrimeric serine-threonine phosphatase which consists of three distinct subunits—the scaffold A subunit (PP2AA), the regulatory B subunit (PP2AB) and the catalytic C subunit (PP2AC), which assembled together into a trimolecular complex28, however the link between PP2A and Th9 cells is not known. The role of PP2A is well characterized in generation and functions of Tregs, as PP2A is essential for IL-2 receptor mediated signaling in Tregs40. Moreover, PP2A in Tregs is also essential for STAT5 mediated functions in Tregs41. Given the shared factor that are required for the generation of Tregs (TGF-β1) and Th9 (TGF-β1 + IL-4), PP2A seems to regulate the pathways that are essential for the generation and functions of Th9 cells. It is known that both IL-2-IL-2R signaling and STAT5 are essential component in the generation and functions of Th9 cells43. IL-2 or STAT5-deficiency leads to the inhibition of generation of Th9 cells43. Our data suggests that, similar to Tregs, PP2A is required for the induction of Th9 cells. It could be possible that inhibition of PP2A in Th9 cells might affect IL-2 and STAT5 mediated signaling which is crucial for the functions of Th9 cells.PP2A has been well described in the differentiation of Th1, Th2, Th17 and Tregs 10, IRF411, HIF1α9, BATF7, GATA36, IRF18 and Foxo15 through PP2A prediction-based interactome. Our data indicated that inhibition of PP2A downregulated the expression of Spi1, Irf4, Hif1α, Batf Gata3, Irf1 and Foxo1 in Th9 cells. This suggests that PP2A interaction with PU.1, IRF4, HIF1α, BATF, GATA3, IRF1 and Foxo1 could be crucial for Th9 cell functions. Our data further demonstrated that siRNA mediated knockdown of PP2A led to diminished IL-9 induction emphasizing the pivotal role of PP2A in Th9 cell differentiation.Our data identified the role of PP2A in Th9 cell differentiation, as pharmacological inhibition of PP2A with okadaic acid or LB-100 led to impairement in the differentiation of Th9 cells. We observed a dose-dependent response of LB-100 in which higher doses (5.0 μM) reduced the proliferation of Th9 cells while an optimal dose of 2 μM was effective in inhibiting PP2A mediated IL-9 induction with lesser reduction in Th9 cell proliferation. We showed a probable interaction of PP2A with the critical transcription factors of Th9 cells such as Spi1+ T cells secreting IFN-γ population in the tumor. Therefore, we report here for the first time that PP2A is essential for the differentiation and anti-tumor functions of Th9 cells.Finally, we validated the in vivo role of PP2A in Th9 cell-mediated anti-tumor immune response. We found that inhibition of PP2A with LB-100 abrogated the anti-tumor response of Th9 cells when these cells were adoptively transferred to B16-OVA tumor bearing mice. This was due to the decreased infiltration of CD8Supplementary file1


Generally, alloying is a traditional and effective way to reduce the lattice thermal conductivity through the mass and strain field fluctuation between host and guest atoms. The cation sites have very few contributions to the band structure around the fermi level; thus, cation substitution may have negligible influence on the electric transport properties. What is more, widespread application of thermoelectric materials not only desires high ZT value but also calls for low-cost and environmentally benign constituent elements. Here, Ba substitution on cation site achieves a sharp reduction in lattice thermal conductivity through enhanced point defects scattering without the obvious sacrifice of high carrier mobility, and thus improves thermoelectric properties. Then, by combining further enhanced phonon scattering caused by isoelectronic substitution of Zn on the Mg site, an extraordinarily low lattice thermal conductivity of 0.51 W m−1 K−1 at 873 K is achieved in (Ca0.75Ba0.25)0.995Na0.005Mg1.95Zn0.05Bi1.98 alloy, approaching the amorphous limit. Such maintenance of high mobility and realization of ultralow lattice thermal conductivity synergistically result in broadly improvement of the quality factor β. Finally, a maximum ZT of 1.25 at 873 K and the corresponding ZTave up to 0.85 from 300 K to 873 K have been obtained for the same composition, meanwhile possessing temperature independent compatibility factor. To our knowledge, the current ZTave exceeds all the reported values in AMg2Bi2-based compounds so far. Furthermore, the low-cost and environment-friendly characteristic plus excellent thermoelectric performance also make the present Zintl phase CaMg2Bi2 more competitive in practical application.CaMg ZT = S2σT/(κE + κL), in which S, T, σ, κE, and κL are Seebeck coefficient, absolute temperature, electrical conductivity, electronic thermal conductivity, and lattice thermal conductivity, respectively [S, σ, and κE hinders the further enhancement of ZT value. Thus, some concepts and strategies including band convergence [κL, the sole independent parameter, are also achieved by introducing nanostructuring [The development of clean energy could help to alleviate the energy crisis , 2. Receectively . Howeververgence , 6, bandvergence , resonanvergence , 9, and vergence , 11 haveucturing , 13, poiucturing –16, dislucturing , 18, latucturing , 20, as ucturing , 22 or eucturing –25.14MnSb11 [1-xYbxZn2Sb2 [3Sb2 [β-Zn4Sb3 [9Zn4Sb9 [0.5Yb0.5)1-xCaxMg2Bi2 [2X2 (1-2-2 type) compounds with CaAl2Si2-type structure deserve intensive attention because of their higher carrier mobility originated from unique crystal structure, where the covalently bonded anionic sheets (M2X2)2- sandwiched between A2+ cations planes  [ZT in excess of 1 [2Bi2  [Zintl phases seem congenital thermoelectric materials for their structural complexity and inherently low thermal conductivity , 27. Som14MnSb11 , Ca1-xYbbxZn2Sb2 , Mg3Sb2 b2 [3Sb2 , β-Zn4Sbβ-Zn4Sb3 , Ca9Zn4S[9Zn4Sb9 , and  . As one ess of 1 –39, whic Yb, Eu) , 41.2Bi2 compound possesses the virtues of environmental friendliness, low cost, and high element abundance compared with the other two containing expensive Yb and Eu. Early studies reported that the peak ZT is only ~0.1 for CaMg2Bi2 synthesized by the traditional melting method, which is far from practical application [+ to replace Ca2+, which eventually causes a mediocre ZT ≈ 0.9 at 873 K for Ca0.995Na0.005Mg2Bi1.98 sample [2Bi2 have been executed to further reduce the κL [ZT value as high as ~1.3 at 873 K in Ca0.6Yb0.2Eu0.2Mg2Bi2 is obtained, the expensiveness of some rare-earth elements makes it difficult to achieve large scale application in some cases [In the Bi-based family, CaMglication . Shuai e8 sample . However8 sample . Attempte the κL , 43. Eveme cases .2Bi2 systems, the element Ba has been verified to be as effective as Yb or Eu in reducing κL [p orbitals dominate the valence band of CaMg2Bi2 [0.995Na0.005Mg2Bi1.98 [κL by constructing point defects, leading to an enhanced thermoelectric properties. Subsequently, Zn doping on Mg site is carried out to further reduce the κL. This equivalent substitution can effectively block phonon transmission without significantly disordering the electrical property. Eventually, the maximum ZT value of ~1.25 at 873 K and the average ZT (ZTave) of ~0.85 between 300 K and 873 K are gained in (Ca0.75Ba0.25)0.995Na0.005Mg1.95Zn0.05Bi1.98 sample. As well, this ZTave is the highest value in p-type AM2X2 Zintl compounds ever reported, and the richness and nontoxic constituent elements are of great practical significance to promote the application of large-scale and high-efficiency thermoelectric modules.In other doped CaMgucing κL . FurtherCaMg2Bi2 , elementg2Bi1.98 , the subx  alloys. The diffraction peaks can be nicely indexed to trigonal CaAl2Si2 structure with the space group Pm1. There is minor impurity phase Bi existing between 25° and 30° on account of the loss of Ca and Mg at elevated temperature. However, it should be noted that the Bragg peaks shift toward lower diffraction angles with increasing Ba fraction as shown in 2+ (1.35 Å) compared to that of Ca2+ (1.0 Å). As anticipated, the calculated lattice parameters a and c linearly increase with increasing Ba content as shown The details of sample preparation and characterization could be found in supporting information. x  alloys. The decreasing trend of σ as temperature increasing represents a typical degenerate semiconducting behavior. Moreover, the electrical conductivity decreases with increasing Ba content during the whole measured temperature range, which could be attributed to the decrease of carrier concentration since the mobility changes slightly with the doping fraction grows as shown in nH and μH as a function of temperature for BaxZny alloys  are shown in nH of Ba-doped samples. The similar phenomenon also appears in Ca1-xYbxMg2Bi2 [1-xCaxZn2Sb2 materials [nH, which is a crucial approach to modulate the S and σ. Worthy of extraordinary attention here is the independence between carrier mobility and Ba doping fraction. The substitution of cation sites may generate minimal impact on the valence band as mentioned above, and thus, the carrier mobility is nearly unchanged with doping content [2Bi2 materials [2Bi2 system, which is critical in developing high thermoelectric performance  alloys. Clearly, the S value increases with increasing Ba fraction, which results from the decreased nH. m∗ = 0.63 me at room temperature under the assumption of a parabolic band and an acoustic phonon scattering [S, the decay of the σ eventually leads to the decline of PF  is determined by SPB model assuming the carriers scattering dominated by acoustic phonon. [x, reaching the minimum value at x = 0.5, and then increases. It should be stressed that the room temperature κL reaches a lowest value of 1.1 W m−1 K−1 for Ba0.5 sample, which is 57% lower than that of undoped sample (2.6 W m−1 K−1). Such low κL is mainly due to additional phonon scattering results from point defect caused by the mass and strain field fluctuation between doping atoms (Ba) and host atoms (Ca). Under the hypothesis that the phonon propagation is only restricted by Umklapp process and point defect scattering, the Debye-Callaway model [κL in diverse systems, such as YbZn2Sb2-xBix [2-xMgxSb2 [1-xSex [x  materials. A systematic description about the model can be found in supporting information. The parameter Γ presents the strength of point defect scattering and is regarded as a product of the multiplication of Γ0 and xi(1 − xi), where Γ0 is a dimensionless parameter obtained utilizing a fitting method and xi is the fractional concentration. The strength of point defect scattering first increases with increasing Ba fraction until x = 0.5 and then decreases  samples are presented in ZT value of 1.2 at 823 K is obtained for Ba0.25 alloy.Combined with the reduced κL reaches 0.57 W m−1 K−1 at 873 K for Ba0.25 sample, this value is still much higher than the theoretical limit (0.35 W m−1 K−1 for Cahill model and 0.14 W m−1 K−1 for Bvk-Pei model) [κL while maintaining the considerable PF is another urgent challenge. One of the interesting characteristics of isoelectronic alloying is that they do not bring the charge disorder but introduce the phonon scattering center due to the mass and strain field fluctuations between the host atoms and the guest atoms [κL without changing the valence band structure [y alloys, which is advantageous for enhancing ZT values.Though the i model) . How to st atoms . In our tructure . Thus, ly  samples is displayed in Figure 2Si2 structure (space group Pm1). It is noted that the lattice parameter increases with Zn content grows up due to the small ionic radius of Zn in comparison with Mg. However, no distinct deviations occur in lattice constant when y exceeds 0.05, which can be attributed to the low solubility limit of Zn in the present compound.Room temperature XRD analysis of BaZnσ and S for BaZny . The slightly variation in the electrical conductivity with increasing Zn fraction can be explained by the almost unchanged carrier concentration and mobility  in (Ca0.75Ba0.25)0.995Na0.005Mg2Bi1.98, its weak carrier scattering effect could be obscured by that caused by Ba doping in (Ca0.75Ba0.25)0.995Na0.005Mg2Bi1.98, leading to an inconspicuous change on mobility. Moreover, the experimental carrier concentration of ~2.5 × 1019 cm−3 for Zn-doped samples is close to the optimal range for maximum PF, as shown in −2 V−1 s−1 is still at a higher level, even compared with other doped CaMg2Bi2 [Figure mobility . Zn dopime group , it is fCaMg2Bi2 , 42–44, PF of BaZny samples increases firstly when y ≤ 0.05 and then decreases (ω) is proportional to the power factor via ω = 1/4((Th − Tc)2/L)PF [PFave of 14.4 μ W cm−1 K−2 in BaZn0.05 sample contributes to a higher output power density compared with the other representative reports [With increasing Zn content, the ecreases . A conclc)2/L)PF . Thus, t reports , 42, 43.κL throughout the measured temperature range due to the enhanced point defects scattering 3/2/κL) was taken as reference to search potential thermoelectric materials [β of BaxZny  is plotted in −4 K m3 V−1 s−1 W−1 is achieved in the x = 0.25, y = 0.05 samples, which is about two times higher than that of the Ba0 sample (31.4 × 10−4 K m3 V−1 s−1 W−1). Thus, with the help of Ba and Zn dual doping, we could realize reduced lattice thermal conductivity without serious compromises in electrical properties. As shown in ZT value of ~1.25 is achieved for BaZn0.05 sample when it is higher than 823 K.In general, for the intrinsic materials metric, quality factor aterials . The rooSnyder and Ursell  proposes another . The sit2Bi2 compounds [ZT value of 1.25 for BaZn0.05 sample catches up with the current highest level, as shown in ZT, a record ZTave of 0.85 is also achieved for the same materials system  play a crucial role in obstructing phonon transport due to the large mass and size difference between the host atom and guest atom. An ultralow κL of 0.51 W m−1 K−1 at 873 K is obtained for (Ca0.75Ba0.25)0.995Na0.005Mg1.95Zn0.05Bi1.98 sample. Further, due to the weakly affected carrier mobility, the quality factor β will be distinctly strengthened. Eventually, the enhanced ZTmax of 1.25 at 873 K and the record ZTave of 0.85 between 300 K and 873 K are also obtained. Last but not least, just this material also displays the temperature independent compatibility factor. Taking into account the practical design of thermoelectric device, high-performance thermoelectric materials composed of cost-effective, environmentally friendly, and plentiful elements are of greatly practical significance for large-scale commercial applications.In this work, p-type CaMg


A significantly (* p < 0.027) lower free asparagine content in the raw material resulted in a lower formation of acrylamide in the final product. The acrylamide content significantly decreased (**** p < 0.0001) after 3 (1100 ± 18 µg kg−1) and 6 (490 ± 7 µg kg−1) months of long-term storage. Only samples stored for 6 months (490 ± 7 µg kg−1) met the EU acrylamide content requirements (<500 µg kg−1) for grain-based coffee substitution products.Acrylamide is assumed to be a potential carcinogen, and reference values have therefore been implemented in EU legislation. Thus, the food industry needs to reduce the acrylamide content in consumer products to the lowest possible value. In this study, roasted rye was evaluated for its suitability as a coffee substitution product with respect to its acrylamide content. The influence of process modifiers, free asparagine content, storage, and rye type on the final content of acrylamide was investigated. Changes in carbohydrate composition and brightness caused by the roasting process were described. Sample analysis was conducted via GC-MS and HPLC-CAD. Existing methods were adapted to roasted rye as the sample matrix. CaCl Acrylamide is an organic compound and belongs to the chemical group of amides . This co3, CaCl2, and asparaginase was discussed [Formed by the Maillard reaction in the presence of reducing sugars and the amino acid asparagine under low-water-content conditions, acrylamide has been of increasing concern for food manufacturers worldwide . The coniscussed ,12. Bein−1 for distribution across the EU, and an even lower level of ≤280 µg kg−1 was required by the Federal Office of Consumer Protection and Food Safety (BVL) in Germany. These restrictions also applied to companies producing coffee substitution products for people suffering from caffeine intolerance or using them due to ecological considerations. Coffee substitutes can be made of local raw materials, including different grain species, such as Taraxacum and Lupinus. After roasting, those products can be prepared with hot water, similar to ground coffee beans. As the taste profile of coffee is mainly dominated by roast aromas, a very similar taste can be achieved by targeted roasting of other raw materials [Hence, model systems have been used to investigate the impact of acrylamide on human health. In the studies conducted, genotoxic and carcinogenic effects were observed ,13. To raterials . Howeveraterials ,16. TherTo assess the toxicity of the final product, analytical methods that offer high sensitivity and low limits of detection and quantitation have been developed over the years to reduce effort in sample preparation and to further increase selectivity and cost efficiency ,18. MostFor this study, CAD was chosen as the most appropriate option for carbohydrate detection . Advanta−1 in acetonitrile), and asparagine (99%), as well as both GC-grade derivatization reagents xanthydrol (≥99%) and N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide (MTBSTFA) (≥99%) with 1% tert-butyldimethylsilyl chloride (TBDMSCI), were obtained from Sigma Aldrich . For pH adjustments, Na2CO3 and acetic acid (80%) were purchased for VWR .Standards of acrylamide (≥99.5%), D3-acrylamide (500 mg L−1) from a Sartorius ultrapure water purification system  was used for sample preparation.Analytical-Grade fructose, glucose, maltose, and sucrose were purchased from Sigma Aldrich. LC-MS-grade acetonitrile and ammonium acetate, as well as ammonia (25%), were obtained from VWR. GC-MS grade ethylacetate, hexane, HCl (37% p.A.) and Carrez I and Carrez II solutions were purchased from VWR. Sample cleanup was performed using J.T. Baker BAKERBOND spe C18 polarplus 1 mL SPE (solid phase extraction) columns . LC-MS-grade water  used were obtained from Arnreiter Mühle GmbH , Plohberger Malz GmbH , and Saatgut Erntegut GmBH . Asparaginase  was purchased as PreventAse (product #23156) from DSM , and CaCl2 was purchased from Solvay .The grain and rye varieties (−1), CaCl2 , Na2CO3  and acetic acid  were diluted in ultrapure water. Grains were soaked in water + modifier for 16 h. Per 400 g sample, 1000 g water + modifier was used. For storage tests, samples were stored at room temperature (25 °C) in their original containers.The modifiers asparaginase  extraction, 100 mg ± 5 mg of ground sample was weighed into a 2 mL centrifugation tube, and 500 µL of hexane, 900 µL of water and 100 µL of D3-AA solution were added. Extraction was performed at 40 °C for 120 min at 1000 rpm in a thermoshaker. After centrifugation at 17,000× −1 HCl and 100 µL of 3.5% xanthydrol solution to 800 µL of the diluted sample. Extraction of xanthyl-AA was achieved by adding 500 µL of ethyl acetate to the sample and further shaking under derivatization conditions for 5 min. The sample was centrifuged afterwards under the conditions mentioned above, and 300 µL of the upper ethyl acetate layer was transferred to a 2 mL GC crimp vial for measurement.Derivatization was performed at 20 °C for 25 min after adding 100 µL of 0.5 moL L−1 AA solution. D3-AA standards without ground samples were prepared separately in triplicate by substitution of 50 µL and 90 µL of the D3-AA solution with water and substitution of 50, 100, 150, 200, 250, and 300 µL of water with a D3-AA solution. Calibration curves were constructed using the peak areas of the obtained 50, 250, 500, 750, 1000, 1250, 1500, 1750, and 2000 µg L−1 AA and D3-AA standards.AA standards without ground samples were prepared separately in triplicate by substitution of 10, 50, 100, 150, 200, 250, 300, 350, and 400 µL of water with a 500 µg L−1 was prepared for use as an external standard.An asparagine solution at 2.5 mg L−1 HCl solution were added. Extraction was performed at 40 °C for 120 min at 1000 rpm in a thermoshaker. After centrifugation at 17,000× g and 25 °C for 5 min, the upper hexane layer was removed. SPE columns were conditioned according to the manufacturer’s instructions. Further sample cleanup was performed via elution of 500 µL of the sample through the SPE column and dilution to 10 mL. For deproteinization, 100 µL of the sample was diluted with 900 µL of acetonitrile and centrifuged under the abovementioned conditions. Consecutively, 500 µL of the sample was transferred into a clean centrifugation tube and dried under a stream of nitrogen.For asparagine extraction, 100 mg ± 5 mg of ground sample was weighed into a 2 mL centrifugation tube, and 500 µL of hexane, 900 µL of water and 100 µL of a 1.0 moL L−1 asparagine solution. Calibration curves were constructed using the peak areas from the silylated 2.5, 5.0, 25.0, 37.5, 50.0, 62.5, 75.0, 87.5, and 100 mg L−1 asparagine standards.After evaporation of solvents, 50 µL of a silylation reagent (MTBSTFA) and 450 µL of acetonitrile were added to the dried tubes. Derivatization was performed at 80 °C for 60 min. After derivatization, the sample was transferred to a 2 mL crimp vial for GC analysis. Asparagine standards were prepared separately without ground samples in triplicate by substitution of 10, 50, 100, 150, 200, 250, 300, 350, and 400 µL of water with a 2.5 mg Lg and at 25 °C for 5 min, 100 µL of supernatant was transferred into a fresh centrifugation tube and vortexed after adding 900 µL of acetonitrile. Precipitants were removed by repeated centrifugation under the abovementioned conditions. For HPLC measurement, 850 µL of supernatant was transferred into a screw-cap vial. Carbohydrate standards were prepared at concentrations of 0.010, 0.050, 0.10, 0.50, 1.0, and 4.0 g L−1 in water. Calibration curves were constructed using the peak areas obtained by measurement. For measurement with charged aerosol detection, no derivatization steps are required.For carbohydrate extraction, 100 mg ± 5 mg of ground sample was weighed into a 2 mL centrifugation tube, and 1000 µL of water was added. Extraction was performed at 40 °C for 120 min at 1000 rpm in a thermoshaker. After centrifugation at 17,000× Whole grain samples were roasted using a drum roaster TKMSX 1 CAFEMINO . Roasted samples were ground using a Perten 3100  laboratory mill. Moist grain was dried using a fluid-bed dryer TG100 .Color measurements of roasted grain were performed by a VeriColor spectro  spectrophotometer. Sample brightness is expressed in lightness values (L *) .Derivatizations and extractions were performed using a thermoshaker. Dry-Matter measurements were performed using an MA 35  moisture analyzer.Acrylamide and asparagine sample analyses were performed on a Thermo Trace 1300 GC equipped with a Thermo TSH100 autosampler coupled to a Thermo ISQ 7000 MS . Carbohydrate experiments were performed on an UltiMate 3000 UHPLC system  equipped with a solvent degasser, a quaternary pump, an autosampler, and a thermostatic column compartment and coupled to a Corona Veo instrument . Data processing was carried out with Chromeleon 7.2.10 software , and the compressed air gas flow rate was automatically regulated and monitored by the CAD device. Data collection was set to 2.0 Hz at a filter constant of 3.6 s. The power function for response and signal correction was set to 1.30.m/z = 206/234 and m/z = 207/237 were used in selected ion mode for identification of xanthyl-acrylamide and D3-xanthyl-acrylamide, respectively. Quantitation of xanthyl-acrylamide and D3-xanthyl-acrylamide was performed in selected ion mode at m/z = 251 and m/z = 254, respectively.Chromatographic separation of acrylamide was achieved using a TRACE TR-5MS  column . The injector port temperature was maintained at 250 °C. The GC column temperature was kept at 60 °C for 1 min and increased from 60 °C to 300 °C in 20 min. During measurements, the transfer line was kept at 280 °C, and the ion source was kept at 300 °C. GC was operated with helium (99.999%) at a constant flow rate of 0.8 mL/min. Each sample was determined in triplicate via splitless injection of 1.3 µL of sample. Fragment ions m/z = 302 was used in selected ion mode for identification of the dimethyl-tert-butylsilyl derivative of asparagine. Quantitation was performed in selected ion mode at m/z = 417.Chromatographic separation of asparagine was achieved using a TRACE TR-5MS  column . The injector port temperature was kept at 300 °C. The GC column temperature was kept at 60 °C for 1 min and increased from 60 °C to 325 °C in 30 min. During measurements, the transfer line was kept at 280 °C, and the ion source was kept at 300 °C. The GC was operated with helium (99.999%) at a constant flow rate of 1.0 mL/min. Each sample was determined in triplicate via splitless injection of 1.0 µL of sample. The fragment ion 4Ac and were adjusted to pH = 8.25 with NH4OH . The buffer components were filtered through a 0.2 µm membrane filter after preparation. The gradient program used is shown in −1) of mobile phase A to prevent sample carryover between runs. The injection volume was 5 µL.Chromatographic separation for carbohydrates was achieved using a WATERS Acquity UPLC BEH amide column  maintained at 40 °C. The autosampler was kept at 25 °C. A guard column  was used to protect the column from particles. Mobile phase A was 85% ACN, and mobile phase B was 60% ACN. All mobile phases contained 10 mM NH2, asparaginase, and pH adjustments), as they potentially impact acrylamide formation; (vi) long-term storage of products at room temperature to validate its influence on acrylamide stability; (vii) the roasting temperature-dependent change in rye flour brightness; and (viii) carbohydrate content and composition before and after roasting.To investigate the applicability of roasted rye a as a coffee substitution product, rye grains of a single type were roasted in a drum roaster. We focused on the following parameters relevant to the formation of acrylamide: (i) filling volume variations in the drum roaster that might also influence the determined concentration of acrylamide; (ii) the asparagine content of rye varieties before and after roasting; (iii) the role of different rye grain types on the formation of acrylamide; (iv) the roasting temperature; (v) modifiers (CaCl−1) and 300 g (1249 ± 15 µg kg−1) showed the lowest standard deviations of all tested filling volumes. No significant impact of filling volume on the formation of acrylamide could be stated. As 400 g of rye per sample was sufficient for analysis, this volume was considered for further experiments.According to the manufacturer’s specifications, the drum roaster used in this study accommodates approximately 1000 g of coffee beans for roasting in one pass. Due to variations in the density of the raw material, it was chosen to test sample weights of 500 ± 200 g for maximum flexibility. The results of acrylamide formation as a function of filling volume are depicted in Regardless of the degree of filling, the rye was roasted for 13 minutes at a constant air temperature of 190 °C. During the roasting process, it was not possible to measure temperature of the individual grains. Therefore, grain brightness was evaluated after roasting .Larger sample volumes were brighter and showed lower acrylamide values than smaller volumes. As the drum roasters heating power was limited, larger filling volumes required more time to heat up compared to smaller volumes and it can be assumed that average grain temperature was lower along the roasting process, respectively.−1) in this study. Only rye variety 1 had a significantly (* p < 0.016) lower asparagine content (560 ± 22 mg kg−1) than the reference samples. Based on the calibration standards, the limit of quantitation (LOQ) was 25 mg kg-1 for the GC method used. None of the roasted rye samples contained asparagine within the range of calibration (<2.5 mg L−1).All four rye varieties used in this study as well as samples of commercially available wheat, spelt, and barley were analyzed for their asparagine content prior to roasting . Rye var−1), as depicted in Next, the influence of four commercially available rye types on acrylamide formation was tested. Rye variety 4 showed the highest tendency for acrylamide formation . The highest acrylamide content (1700 ± 26 µg kg−1) was observed for samples roasted at 150 °C. The water content after the roasting process ranged from 1.85–2.39%.The results depicted in p < 0.05) in samples treated with 300 mg∙L−1 asparaginase (1000 ± 12 µg kg−1). Untreated (890 ± 35 µg kg−1) and 200 mg∙L−1 (890 ± 16 µg kg−1) samples showed almost equal acrylamide values. As a control experiment, the acrylamide content was determined at reduced roasting times. As expected, samples treated with 100 mg∙L−1 asparaginase showed an increased acrylamide content with increased roasting time.The role of modifiers in acrylamide formation was investigated. The results for asparaginase-treated samples a were co2 (p < 0.028) acrylamide content compared to that of the control sample. During roasting, the acrylamide content first increased  and then decreased  in samples treated with 0.2 moL L−1 CaCl2. The water content after the roasting process ranged from 0.17–4.02%.Samples treated with CaCl2 b showed −1). In samples adjusted to pH = 8.0 (1000 ± 17 µg kg−1) and pH = 9.0 (940 ± 18 µg kg−1), the increase in acrylamide content was lower than that in acidic samples adjusted to pH = 5.0 (1182 ± 28 µg kg−1) and pH = 6.0 (1127 ± 11 µg kg−1). The acidic samples seemed to be more prone to acrylamide formation than the alkaline samples.Samples soaked in pH-adjusted water c were cop < 0.0001) after 3 (1100 ± 18 µg kg−1) and 6 months (490 ± 7 µg kg−1) of storage compared to that acrylamide content in the fresh samples (1400 ± 11 µg kg−1). The water content increased from 1.85–2.59% to 2.41–3.12% over 6 months.The influence of storage duration on acrylamide content in roasted rye was tested. Degradation of acrylamide  proved tR2 = 0.9194) was observed for all samples in The brightness decrease caused by roasting (−1) and fructose (0.44 ± 0.004 g kg−1) contents increased , whereas the glucose (0.83 ± 0.007 g kg−1) and sucrose (0.33 ± 0.065 g kg−1) contents decreased  after roasting.The contents of fructose, glucose, sucrose, and maltose in rye flour before and after roasting were determined . The malThe different batches showed the highest variation in maltose content (27%) and the lowest variation in sucrose content (7%). The content of reducing carbohydrates involved in acrylamide formation, fructose (10%), and glucose (13%), varied remarkably between rye batches. The results indicate that maltose can be formed by heat treatment of glucose, whereas fructose is released from polymers or formed from sucrose.In this study, we investigated the efficacy of modifiers, substances that were added before or during the roasting process, to inhibit acrylamide formation. Therefore, a rye variety with a high initial acrylamide content after roasting (rye variety 4) was chosen. We found that the free asparagine content  in the rWe also studied the influence of the roasting temperature  on acryl2, acetic acid, and Na2CO3 were tested as modifiers and compared to results from other studies performed on different kinds of foodstuffs [2 had no effect on acrylamide content in the final product  showed a linear correlation . StabiliImportantly, consumer acceptance is strongly correlated with the brightness value, as higher roasting temperatures increase typical roast flavors demanded by the customer. Rye roasted at temperatures above 170 °C meets the requirements of brightness values <65. Therefore, future studies should focus on the reduction of acrylamide content in rye roasted above those temperatures to further increase consumer acceptance.The acrylamide content in roasted rye was higher than that in roasted coffee beans. The modifiers used in this study did not decrease acrylamide formation. However, long-term storage and low-temperature roasting can be used to reduce the acrylamide content in the final product. The brightness of the product is directly related to the roasting temperature, which also changes the carbohydrate profile.


AfCel6A) and lytic polysaccharide monooxygenase LPMO (AfAA9_B) from Aspergillus fumigatus after they were expressed in Pichia pastoris and purified. The biochemical parameters suggested that the enzymes were stable; the optimal temperature was ~60 °C. Further characterization revealed high turnover numbers . Surprisingly, when combined, AfCel6A and AfAA9_B did not act synergistically. AfCel6A and AfAA9_B association inhibited AfCel6A activity, an outcome that needs to be further investigated. However, AfCel6A or AfAA9_B addition boosted the enzymatic saccharification activity of a cellulase cocktail and the activity of cellulase Af-EGL7. Enzymatic cocktail supplementation with AfCel6A or AfAA9_B boosted the yield of fermentable sugars from complex substrates, especially sugarcane exploded bagasse, by up to 95%. The synergism between the cellulase cocktail and AfAA9_B was enzyme- and substrate-specific, which suggests a specific enzymatic cocktail for each biomass by up to 95%. The synergism between the cellulase cocktail and AfAA9_B was enzyme- and substrate-specific, which suggests a specific enzymatic cocktail for each biomass.Cellulose is the most abundant polysaccharide in lignocellulosic biomass, where it is interlinked with lignin and hemicellulose. Bioethanol can be produced from biomass. Since breaking down biomass is difficult, cellulose-active enzymes secreted by filamentous fungi play an important role in degrading recalcitrant lignocellulosic biomass. We characterized a cellobiohydrolase ( In this scenario, lignocellulosic residues stand out as a new generation of renewable energy sources, including second-generation (2G) ethanol , where θ is the ellipticity value originally given by equipment (millidegrees), MRW is the enzyme mean residual weight, d is the optical path (cm), and c is the enzyme concentration (mg mL−1). All the secondary structures of the enzymes were predicted by using the BeStSel web server [Circular dichroism (CD) spectra of the enzymes were obtained between 190 and 250 nm (far-UV) on a JASCO-810 spectropolarimeter; quartz cuvettes with optical path of 0.1 cm were employed. b server , and theb server  and DiscAfAA9_B activity was analyzed as reported by Breslmayr et al. (2018) [2O2, and recombinant purified AfAA9_B in 50 mM sodium phosphate buffer (pH 8.0). For the blank, the enzyme was denatured by incubation at 99 °C for 30 min before the reaction mixture was added. After 5 min at 30 °C, absorbance was read at 469 nm to calculate the LPMO peroxidase activity.Purified . (2018) . The assAfCel6A activity was determined by measuring reducing sugars from the reaction by the 3,5-dinitrosalicylic acid (DNS) method [w/v) in 50 mM sodium phosphate buffer (pH 6.0) was incubated at 55 °C for 30–45 min. The enzymatic action was stopped by adding an equal volume of the DNS reagent. The mixture was boiled for 5 min and cooled, and absorbance was measured at 540 nm. One unit of AfCel6A was defined as the amount of enzyme that released 1 µmol of reducing sugar from the substrate per minute. Each assay was carried out in triplicate. Enzyme concentration was determined by the Greenberg method [) method . Brieflyg method .AfAA9_B activity was measured at pH ranging from 4.0 to 8.0 in McIlvaine buffer (citric acid-Na2HPO4) and at pH 9.0 and 10.0 in 100 mM Glycine-NaOH buffer at 30 °C. The relative activity was calculated with respect to the maximum activity of 100%; the aforementioned method was followed. The pH stability was estimated by measuring the residual enzymatic activity after the enzyme was incubated without substrate in the aforementioned buffers at pH ranging from 3.0 to 10.0 and 4 °C for up to 72 h. To determine the AfAA9_B thermal stability, the enzyme was preincubated without substrate at 50 and 60 °C for up to 72 h. To measure the residual activity, the enzymatic activity without preincubation was considered 100%.The optimal pH for AfCel6A activity was measured from 3.0 to 8.0 in McIlvaine buffer (citric acid-Na2HPO4) at 55 °C. The optimal temperature was examined between 40 and 80 °C. The relative activity was calculated with respect to the maximum exhibited activity of 100%; the aforementioned method was followed.The optimal pH for AfCel6A pH stability was estimated by measuring the residual enzymatic activity under standard conditions after the enzyme was incubated without substrate in Mcllvaine (citrate–phosphate) buffers pH 3.0–8.0 and in 100 mM Glycine-NaOH buffers pH 9.0 and 10.0 at 4 °C for up to 72 h. To determine the AfCel6A thermal stability, the enzyme was preincubated without substrate at temperatures ranging from 50 to 90 °C for different times. To measure the residual activity, the enzymatic activity without preincubation was considered 100%.The AfAA9_B and AfCel6A was determined by adding Mn2+, Co2+, Ca2+, Fe2+, Zn2+, Mg2+, Cu2+, NH4+, K+, or Ag+ at a final concentration of 5 mM to the reaction mixture. The effects of EDTA, SDS, Tween 20, Triton X-100, SLS, β-mercaptoethanol, DTT, and DMSO were also tested. For AfCel6A, the effect of ascorbic acid addition was also evaluated. Control reactions (100% activity) were performed without any additive. The relative activity was estimated as compared to the controls.How various metal ions affected AfAA9_B and AfCel6A were determined in the presence of increasing concentrations of both sugars by using the chromogenic substrates 2,6-DMP and 4-nitrophenyl β-D-cellobioside , respectively.The glucose (10–250 mM) and cellobiose (up to 100 mM) effects on the activity of AfAA9_B kinetic parameters  were determined for the substrate 2,6-DMP (0.1 to 10 mM) and the co-substrate H2O2 (1 to 500 µM). The reactions were performed in 50 mM sodium phosphate buffer (pH 6.0) and 100 mM glycine-NaOH buffer (pH 9.0) at 50 °C. The parameters were calculated by Michaelis–Menten nonlinear regression.The AfCel6A kinetic parameters were determined when CM-Cellulose (0.5–30 mg mL−1) was used as substrate. The reactions were performed in 50 mM sodium phosphate buffer (pH 6.0) as previously described. The parameters were calculated by the Michaelis-Menten nonlinear regression graphical method.The AfAA9_B and AfCel6A enzymatic assays were carried out concomitantly with the recombinant endoglucanase Af-EGL7 as previously described [Af-EGL7 to 50 µg of AfAA9_B (1:50) or 10 µg of AfCel6A (1:10) per gram of substrate. The reaction mixtures consisted of CM-Cellulose (1% (w/v)) in 50 mM sodium phosphate buffer (pH 6.0) containing 1 mM ascorbic acid in a final volume of 1 mL. The reactions were performed in a thermomixer (Eppendorf) at 50 °C and 1000 rpm for 4, 8, or 24 h.escribed . The assAfAA9_B and AfCel6A were combined at different concentration proportions , where the minimum and maximum enzyme loading corresponded to 5 and 50 µg of added enzyme per gram of CM-Cellulose, respectively.In the same way, Af-EGL7 concentration was 1 µg g−1, the concentrations of both AfAA9_B and AfCel6A were 10 µg per gram of CM-Cellulose, generating the ratio 1:10:10. The reactions were carried out as described above.Finally, the effect of the simultaneous association of the three recombinant enzymes on the degradation of CM-Cellulose was evaluated. While the The degradation efficiencies were assessed by estimating the released reducing sugars by the DNS method. The reported results represent the mean ± SD calculated from at least three experimental replicates.AfAA9_B and AfCel6A synergistic activity of during enzymatic hydrolysis was investigated in combination with Celluclast® 1.5L, a commercial cellulase cocktail from Trichoderma reesei.® 1.5L cocktail was associated with 50 µg of AfAA9_B (ratio 1:10) or 5 µg of AfCel6A (ratio 1:1) per gram of CM-Cellulose (1% (w/v) in 50 mM sodium phosphate buffer (pH 6.0) containing 1 mM ascorbic acid. The reactions were conducted at 1000 rpm and 50 °C for up to 24 h in a final volume of 1 mL.To this end, 0.05 FPU of CelluclastA. fumigatus on the degradation of CM-Cellulose was also evaluated. While the Celluclast® 1.5 L cocktail loading was fixed at 0.05 FPU g−1, the AfCel6A and AfAA9_B concentrations were 5 and 0.5 µg of enzyme added per gram of CM-Cellulose, respectively. The reactions were carried out as described above.The effect of the simultaneous association between commercial cellulases and the two recombinant enzymes from The percent hydrolysis yields were determined by estimating the released reducing sugars by the DNS method . The repw/v) of one of the following biomasses: SEB (sugarcane exploded bagasse), rice straw, or corncob.Enzymatic hydrolyses of some agro-industrial residues were carried out as described by Bernardi et al. (2019) with some modifications . SaccharAf-EGL7 (18 µg g−1) was combined with AfAA9_B (900 µg g−1) or AfCel6A (180 µg g−1). Similarly, a fixed concentration of Celluclast 1.5L cocktail (0.9 FPU g−1) was associated with AfAA9_B (900 µg g−1) or AfCel6A (90 µg g−1). The reactions were conducted at 1000 rpm and 50 °C for up to 48 h in a final volume of 1 mL. DNS was added to stop the reactions and to measure the released reducing sugars. The reported results represent the mean ± SD calculated from at least three experimental replicates.Different associations between the enzymes were used during biomass saccharifications. All the data are the mean of at least three independent experiments and show consistent results.Aspergillus fumigatus were characterized after they were expressed in P. pastoris. Supplementation of a cellulase cocktail with both enzymes improved the yield of saccharification of different biomasses, especially SEB. However, AfAA9_B did not have a positive effect on AfCel6A activity. On the other hand, AfAA9_B acted synergistically with endoglucanase Af-EGL7. These different synergistic effects are important to understand the action of LPMOs with cellulases and would help to design new commercial enzymatic cocktails. Considering the reduction of costs in lignocellulose conversion, we can conclude that supplementation of Celluclast® 1.5L with AfCel6A or AfAA9_B suffices to increase the hydrolytic activity, so the composition of cellulase cocktails may need to be reconsidered.Novel cellobiohydrolase and LPMO from


UPLC-MS/MS was used to detect the probes and their metabolites in rat plasma and rat liver microsomes. The in vitro results revealed that the half-maximal inhibitory concentration values of bupropion and tolbutamide in RLMs were 8.79 and 20.17 μM, respectively, indicating that poziotinib showed varying degrees of inhibition toward cyp2b1 and cyp2c11. Poziotinib was a competitive inhibitor of cyp2b1 and cyp2c11, with Ki values of 16.18 and 17.66 μM, respectively. No time- or concentration-dependence of inhibition by poziotinib was observed toward cyp2b1 and cyp2c11 in RLMs. Additionally, no obvious inhibitory effects were observed on the activity of cyp1a2, cyp2d1, cyp2e1, and cyp3a1/2. In vivo analysis revealed that bupropion, tolbutamide, phenacetin, and chlorzoxazone showed significantly different pharmacokinetic parameters after administration (p < 0.05); there was no significant difference in the pharmacokinetic parameters of dextromethorphan and midazolam. These results show that poziotinib inhibited cyp2b1 and cyp2c11, but induced cyp1a2 and cyp2e1 in rats. Thus, poziotinib inhibited cyp2b1 and cyp2c11 activity in rats, suggesting the possibility of interactions between poziotinib and these CYP substrates and the need for caution when combining them in clinical settings.Poziotinib is an orally active, irreversible, pan-HER tyrosine kinase inhibitor used to treat non-small cell lung cancer, breast cancer, and gastric cancer. Poziotinib is currently under clinical investigation, and understanding its drug-drug interactions is extremely important for its future development and clinical application. The cocktail method is most suitable for evaluating the activity of cytochrome P450 enzymes (CYPs). As poziotinib is partially metabolized by CYPs, cocktail probes are used to study the interaction between drugs metabolized by each CYP subtype. Midazolam, bupropion, dextromethorphan, tolbutamide, chlorzoxazone, phenacetin, and their metabolites were used to examine the effects of poziotinib on the activity of cyp1a2, 2b1, 2d1, 2c11, 2e1, and 3a1/2, respectively. The  Drug-drug interaction (DDI) occurs during the co-administration of two or more drugs and may increase toxicity or weaken the therapeutic effect of the drugs, leading to the occurrence of serious clinical complications such as adverse drug reactions or treatment failure . DDI is CYP is a superfamily of oxygenases with mixed functions that are involved in the metabolism of endogenous and exogenous substances and are abundantly present in the liver and intestines . CYPs arCYP2C is the second most abundant isoenzyme subfamily. Its main isoenzymes are CYP2C9 and CYP2C19 can metabolize ∼25% of all drugs, of which CYP2C9 metabolizes 16% of all drugs, e.g., oral anticoagulants, non-steroidal anti-inflammatory drugs, various fatty acids, and steroid hormones . CYP1A2 The cocktail method, involving the simultaneous use of several probe substrates, has been used to investigate DDIs and examine changes in CYP activity in drug metabolism . The FDAEGFR and HER2 exon 20 mutations (Poziotinib (HM781-36B) is a new, orally active, irreversible pan-HER tyrosine kinase inhibitor . Poziotiutations . Analyseutations . This evin vivo and in vitro. The results of this study will help to understand the potential drug interactions of poziotinib.It is extremely important to select the appropriate probe substrate for each CYP450 enzyme. Studies reporting the cocktail probe approach have used phenacetin for CYP1A2, bupropion for CYP2B6, tolbutamide for CYP2C9, dextromethorphan for CYP2D6, chlorzoxazone for CYP2E1, and midazolam for CYP3A4 (Poziotinib (purity > 98%) was purchased from the Beijing Sunflower and Technology Development Co. Ltd. . Bupropion hydrochloride (purity > 98%) and phenacetin (purity > 99%) were purchased from TCI Chemicals . Tolbutamide (purity > 97.5%) and chlorzoxazone (purity > 98%) were purchased from J&K Chemical Ltd. (Shanghai China). α-Naphthoflavone (purity > 98%), Ticlopidine (purity > 98%), sulfaphenazole (purity > 98%), quinidine (purity > 98%), sodium diethyldithiocarbamatre (purity > 97%), ketoconazole (purity > 99%) were purchased from Aladdin. Midazolam injection was obtained from Nhwa Pharma. Co.  and NADPH from Roche . The internal standard (IS) of diazepam (purity > 98%) was purchased from the Tianjin KingYork Pharmaceutical Co. Ltd. . Chromatographic grade methanol and acetonitrile were purchased from Merck Co. Ltd. . Rat liver microsomes (RLMs) were prepared in our laboratory . All othSamples were analyzed by a UPLC-MS/MS system equipped with a triple quadrupole mass spectrometer  and Acquity UPLC BEH C18 column  at 40°C. The mobile phase consisted of acetonitrile (A) and 0.1% formic acid (B) with a linear gradient elution at 0.4 ml/min for 3 min. The elution procedure was as follows: 0.0–0.6 min (rapidly rising from 10 to 50% A), 0.6–1 min (increasing from 50 to 80% A), 1–2 min (increasing from 80 to 95% A), 2–2.5 min (maintained at 95% A), and 2.5–2.6 min (reduced to 10% A).Mass spectrometer contained a triple quadrupole and an ESI source. The optimized parameters of multiple reaction monitoring detection included a source temperature of 150°C and desolvation temperature of 500°C. The MS conditions, chemical structure, and chromatograms for the probe drugs and IS are presented in 50) of poziotinib was determined with all of the CYPs. Phenacetin, bupropion, tolbutamide, dextromethorphan, chlorzoxazone, and midazolam  were mixed to form a cocktail of probe substrates for measuring CYP450 activity. To this cocktail, 1 mg/ml of RLMs, 100 mM potassium phosphate buffer (pH 7.4), poziotinib  were added to obtain a final volume of 200 μL. After pre-incubation of this mixture for 5 min in a shaking water bath at 37°C, 1 mM NADPH was added to start the reaction. The reactions were allowed to proceed for 30 min, after which 200 μL of acetonitrile and 20 μL of IS were added to terminate the reactions. The mixtures were vortexed for 30 s and centrifuged again at 13,000 rpm for 5 min. Then, 2 μL of the supernatant was injected into the UPLC-MS/MS system for analysis.The cocktail method was employed to analyze the effects of poziotinib on cyp1a2, cyp2b1, cyp2c11, cyp2d1, cyp2e1, and cyp3a1/2. First, 100 μM poziotinib was used to test its direct inhibitory effects RLMs. Then half-maximal inhibitory concentration , and NADPH was pre-incubated at 37°C for 0, 5, 10, and 20 min; Methanol instead of poziotinib was performed as control. Then 20 μM bupropion or 100 μM tolbutamide was added and incubated at 37°C for 30 min. The reactions were carried out and analyzed in triplicate as described in 2.3.ad libitum for 2 weeks for acclimation. All of the experimental procedures followed the guidelines for the care and use of laboratory animals and were approved (No. wydw 2019-650) by the Animal Experimental Ethical Inspection of Laboratory Animal Center, Wenzhou Medical University.Male Sprague-Dawley rats were supplied by the Animal Experimental Center of the Wenzhou Medical University . Animals were kept under standard conditions with a 12-h day-night cycle and were provided water and food Twelve male Sprague-Dawley rats (230 ± 20 g) were randomly divided into treated and control groups, with 6 in each group. Dose of probe substrate mixture  was dissolved in 0.5% CMC-Na solution: 10 mg/kg, Toluamide: 1 mg/kg. After 7 days of continuous oral administration of poziotinib  to the experimental group, the two group rats were treated with an oral gavage of probe substrate mixture. After 0.083, 0.25, 0.5, 1, 2, 3, 4, 6, 8, 12, and 24 h following administration of poziotinib, 300 μL blood was collected from the tail vein of each rat, transferred to a heparinized centrifuge tube, and immediately centrifuged at 4,000 rpm for 10 min. The supernatant was mixed with 200 μL of acetonitrile and 20 μL of 0.5 μg/ml diazepam (IS), vortexed for 30 s, centrifuged at 13,000 rpm for 5 min, and the supernatant was injected into the UPLC-MS/MS system for analysis.p < 0.05 was considered statistically significant. GraphPad  was used to calculate IC50 and plot the plasma concentration-time curves and inhibition plots .Pharmacokinetic parameters were calculated using DAS software (Version 3.2.8). SPSS software  was used to analyze the significance of differences between the main pharmacokinetic parameters. 50 values of the six probe substrates  from those of the control group ((0–t), AUC (0–∞), and Cmax values of the treated group were significantly reduced by 56.08, 55.81, and 48.06%, respectively. In addition, t1/2, Vz/F, and CLz/F values were significantly increased by 45.45, 203.82, and 116.85%, respectively. The higher AUC and Cmax values and lower Tmax values of the control group indicated that poziotinib induces cyp1a2 activity, thus accelerating the metabolism of phenacetin and reducing its plasma drug concentration in rats.The pharmacokinetic parameters of phenacetin were used to describe the inhibitory effects of poziotinib on cyp1a2 activity. The area under the plasma concentration-time curve of phenacetin  in the tol group . Compare(0–t), AUC(0–∞), MRT(0–t), and Tmax values in the treated group were 1.39, 1.46, 1.41, and 1.63-fold higher than those of the control group, respectively, whereas the CLz/F and Vz/F values were 1.47 and 1.04-fold lower than those of the control group, respectively  and AUC (0–∞) values were significantly higher in the treated group than in the control group (p < 0.05), increasing by 45.87 and 51.60%, respectively, whereas CLz/F was significantly lower than control group by 50% , AUC(0–∞), and Cmax values of the treated group were significantly reduced to 1.95, 1.94 and 2.51-fold that of the control group, respectively. The MRT (0–t), MRT (0–∞), Vz/F, and CLz/F of the treated group were 1.60, 1.56, 1.75, and 1.96-fold higher than those of the control group  > IC50 (tolbutamide) > IC50 (dextromethorphan) > IC50 (phenacetin) > IC50 (chlorzoxazone) > IC50 (midazolam). According to literature, IC50 < 1 μM is considered to be strong inhibition, 1 μM < IC50 < 10 μM moderate inhibition, and IC50 > 10 μM weak inhibition (50 = 8.79 μM) and CYP2C11 (IC50 = 20.17 μM), respectively, whereas its inhibitory effect on the other four CYPs was not obvious . A more detailed investigation of the inhibitory effect of poziotinib on cyp2b1 and 2c11 revealed that the inhibition of both cyp2b1 and 2c11 by poziotinib was competitive, with Ki values of 16.18 and 17.66 μM, respectively.The results of hibition . Our resAs the approved EGFR inhibitor, poziotinib is a quinazoline derivative, same as afatinib, erlotinib, gefitinib, dacomitinib. The above quinazoline derivatives are all metabolized by CYP 2D6 and CYP 3A4, however, they have different impacts on CYPs activity. Afatinib has no inhibition or induction on cytochrome P450 (CYP) enzymes . ErlotinThis competitive inhibition indicates that the inhibitor, poziotinib, and the enzyme substrate compete at the binding site, thereby interfering with the binding of the substrate to the enzyme and reducing enzymatic activity. In addition, we did not observe any time- and concentration-dependent inhibitory effects of poziotinib on cyp2b1 and 2c11 in RLMs, which implies that the activity of these enzymes did not change over time during the incubation period. These results indicate that cyp2b1 and cyp2c11 were reversibly inhibited by poziotinib. Therefore, attention should be paid to the combined use of poziotinib and drugs to look for possible interactions of poziotinib with drugs that are metabolized by cyp2b1 or cyp2c11 to prevent the occurrence of serious adverse reactions.(0–t) and CLz/F values of the treated group were 1.40 and 0.68-fold that of the control group values, respectively. Similarly, for cyp2c11 and tolbutamide, the AUC(0–t) and CLz/F values of the treated group were 1.43 and 0.5-fold that of the control group values, respectively. These results indicate that the activity of cyp2b1 and 2c11 was inhibited by poziotinib, which resulted in increased systemic exposure of bupropion and tolbutamide in rats and slower clearance rates. This was consistent with the results of the in vitro incubation with RLMs, confirming the inhibitory effect of poziotinib on the activity of cyp2b1 and 2c11.We compared pharmacokinetic parameters of the six CYP probe substrates as a result of their interaction with poziotinib in rats. For cyp2b1 and bupropion, the AUCmax values in the treated groups were significantly lower than those in the respective control groups, whereas CLz/F values were much higher than those in the control groups. Thus, poziotinib accelerated the elimination rates and reduced the plasma concentrations of phenacetin and chlorzoxazone in rats. However, our in vitro results did not reveal any inhibitory effect of poziotinib on cyp1a2 and 2e1. This could imply that the observed induction in rats may be caused by gene regulation, which increased protein synthesis and accelerated the metabolism of the probe substrates; however, further research is needed to confirm this hypothesis (in vivo or in vitro.This inhibition could be attributed to the direct binding of poziotinib to the substrate binding site in the enzymes, leading to a decrease in the metabolic activity of these two enzymes. Another study had postulated that long-term use of poziotinib may affect the expression of metabolic enzymes . In addipothesis . PoziotiIn conclusion, this study shows that poziotinib competitively and reversibly inhibited the metabolism of cyp2b1 and cyp2c11 substrates to varying degrees without any time- or concentration dependence. Poziotinib had no obvious inhibitory effect on the other four CYPs. The results of this study will help in further study of poziotinib in humans and draw attention to its potential DDIs when combined with other drugs metabolized by cyp2b1 and cyp2c11 to enable clinically effective and safe use of poziotinib.


Two plant βCA isoforms are targeted to the chloroplast and represent abundant proteins in the range of >1% of chloroplast protein. While their function in gas exchange and photosynthesis is well-characterized in carbon concentrating mechanisms of cyanobacteria and plants with C4-photosynthesis, their function in plants with C3-photosynthesis is less clear. The presence of conserved and surface-exposed cysteinyl residues in the βCA-structure urged to the question whether βCA is subject to redox regulation. Activity measurements revealed reductive activation of βCA1, whereas oxidized βCA1 was inactive. Mutation of cysteinyl residues decreased βCA1 activity, in particular C280S, C167S, C230S, and C257S. High concentrations of dithiothreitol or low amounts of reduced thioredoxins (TRXs) activated oxidized βCA1. TRX-y1 and TRX-y2 most efficiently activated βCA1, followed by TRX-f1 and f2 and NADPH-dependent TRX reductase C (NTRC). High light irradiation did not enhance βCA activity in wildtype Arabidopsis, but surprisingly in βca1 knockout plants, indicating light-dependent regulation. The results assign a role of βCA within the thiol redox regulatory network of the chloroplast.β-carbonic anhydrases (βCA) accelerate the equilibrium formation between CO Since the beginning of industrialization in the 18th century, the CO2 concentration is exponentially increasing and has reached 417 ppm in May 2020 . Despite this increase, the current CO2 concentration is still limiting for C3 photosynthesis where the lyase ribulose-1,5-bisphosphate carboxylase oxygenase (RubisCO) carboxylates ribose-1,5-bisphosphate. This reaction is limited by the low KM(CO2) of RubisCO in the range of 10–12 µM, and the competing reaction of RuBP oxygenation with a KM(O2) of about 600 µM [2 fixation rates of C3-plants tentatively increase threefold, if the ambient CO2 concentration increases to saturation [The atmospheric CO2 limitation of photosynthetic CO2 assimilation and plant yield formation was the driving force for evolution of carbon concentrating mechanisms (CCM). Cyanobacteria express carboxysomes as a bacterial microcompartment in order to increase the CO2 concentration at the active site of RubisCO [3−, but impermeable for CO2. CA generates a high CO2 concentration in the carboxysome. Shell breakage may cause carboxysome loss in cyanobacteria [The CO RubisCO . Aggregabacteria .2 concentration mechanism where HCO3− is fixed by phospho-enol-pyruvate carboxylase (PEPC) in the mesophyll to produce oxaloacetate, which is reduced to malic acid with NADH + H+ as an electron donor. β-carbonic anhydrase (βCA) catalyzes the hydration of CO2 to HCO3−, which serves as substrate for PEPC. After transport of suitable metabolites like malic acid to the bundle sheath, CO2 is released in the bundle sheath by decarboxylation. Mathematical modeling of C4 photosynthesis reveals that βCA activity influences the leakiness of CO2 from bundle sheath cells. Sufficient βCA activity assures a low leakiness of CO2 out of the bundle sheath. This ensures the needed CO2 saturation of the Calvin–Benson cycle (CBC) [C4 plants like maize employ a COle (CBC) .2 + H2O ↔ H+ + HCO3− with turnover numbers of >106 s−1. The high turnover number reveals a diffusion-limited CA activity. CAs are grouped into eight classes namely α, β, γ, δ, ζ, η, θ, and ι [Arabidopsis thaliana codes for 19 CA genes, namely 8 αCAs, 6 βCAs, 3 γCAs, and 2 γ-like CAs [CAs are metalloenzymes catalyzing the mutual conversion of COlike CAs .βca1 knockout Arabidopsis plants display lower survival rates during germination and seedling growth. Growth in elevated CO2 rescues the wildtype phenotype [βca1-βca4 lost its ability to close its stomata in response to high ambient CO2 [3− on stomatal aperture could be traced to the activation of the slow anion channel associated 1 (SLAC1) via the protein kinase open stomata 1 (OST1) [βCAs display a catalytic Zn-ion coordinated by two Cys and one His residue . βCAs lohenotype . Adult pient CO2 . This ef1 (OST1) .Phaeodactylum tricornutum. These results indicate that also Arabidopsis thaliana βCA contain redox-sensitive Cys thiols. Thiol redox regulation is a prominent mechanism to control chloroplast metabolism, in particular, the activity of the CBC. A profound understanding of the redox regulatory network has emerged. It consists of TRX and GRX as redox transmitters, many polypeptides as regulated redox targets, reactive species as final electron acceptors, and thiol peroxidases as redox sensors [βCAs were frequently recovered in redox proteomics approaches. Thus, Balmer et al.  pulled o sensors ,18.Arabidopsis thaliana by oxidation, we sought for the chloroplast reductant able to activate oxidized βCA. In this work, we tested the impacts of typical thioredoxins  or atypical multidomain thioredoxin (CDSP32) or the atypical thioredoxin reductase (NTRC) on the βCA1 activity and conformation (oligomeric state). In addition, site-directed mutation of exposed Cys residues helped identifying involved thiols. Finally, to investigate the role of βCA1 in planta, activity was determined in leaf extracts from wildtype (WT) and βca1 knock-out mutants exposed to different light intensities.For this reason, this work was designed to address the open question whether chloroplast βCA is subject to thiol redox regulation. Following establishment of the inhibition of βCA1 from βCA1 was cloned into pET28a using the primer as depicted in E. coli NiCo21 (DE3) cells were transformed with plasmid DNA. After induction with 100 µM isopropyl-β-D-thiogalactopyranoside, recombinant protein expression was carried out for 4 h at 37 °C and 170 rpm. Cells were harvested by centrifugation . Pelleted cells were resuspended in lysis buffer , disrupted by sonication  and centrifuged at 12,000× g and 4 °C for 60 min. After passage through a chitin column , the lysate was incubated with Roti ® Garose-His/Ni NTA-beads  under gentle shaking at 4 °C for 60 min. After exhaustive washing with washing buffer I  and washing buffer II , the his-tagged protein was eluted using the elution buffer . The eluted proteins were dialyzed over night at 4 °C against 40 mM K-phosphate pH 7.2. βCA1 and the Cys variants were dialyzed over night at 4 °C against 50 mM Na-phosphate pH 8. Prior to usage, the proteins were centrifuged for 5 min at 13,000 rpm to remove denatured protein. Protein concentrations were determined spectrophotometrically using the protein-specific molar extinction coefficient at 280 nm calculated with the Expasy ProtParam tool [2 dmol−1 per residue).Circular dichroism (CD) spectra in the far-UV range were recorded in a Chirascan-plus spectropolarimeter equipped with a Peltier system . CD spectra were obtained with 50 µM βCA1 in a flat quartz cell (path length 0.01 cm) at 273 K using a scan rate of 0.5 nm/s, in the far-UV range (180 to 260 nm), and are the average of three successive measurements. The spectrum of a buffer blank was subtracted from the raw CD data, and the result was expressed as molar ellipticity per amino acid . Prior to analysis, all samples were centrifuged at 21,130× g at 4 °C for 30 min to eliminate disturbing particles with a hydrodynamic diameter greater than 1 µm that could interfere with the measure. A 15 µL supernatant volume was transferred into a quartz cuvette of 15 µL and the particles size measured in triplicate at 25 °C with a light diffusion angle of 173°. The data were collected in automatic mode and analyzed using the associated software DTS v4.2. Data were expressed either in intensity or after mathematical treatment using Mie theory [βCA1 size was determined by photon correlation spectroscopy on a dynamic light scattering (DLS) Zetasizer Nano-S model . Purified βCA1 was analyzed by DLS at the final concentration of 320 µM either after addition of 20 mM DTT or in the presence of 500 µM H2O2 for 30 min at room temperature. One part of the oxidized protein was loaded on the SEC column or incubated with reduced TRX-f1 for 30 min at room temperature and then loaded on the SEC column.Size exclusion chromatography (SEC) was done using the HiPrep 16/60 Sephacryl S200 HR column . Protein thiols were reduced with 50 mM DTT at room temperature for 30 min. A PD10 column was used to desalt the protein. Part of the protein was loaded on the SEC column. The other part was oxidized with 100 µM H6-tagged βCA1 was pretreated with indicated DTT concentrations for 30 min at room temperature. For the reoxidizing experiment, reduced βCA1 was desalted using Zeba Spin Desalting Columns  prior to oxidation with indicated substances. The βCA1 protein was added to 2 mL 20 mM Tris-HCl (pH 8.5 at 5 °C) with 15 µg/mL phenolred  at a final concentration of 50 nM βCA1. Then, 1.33 mL of ice-cold CO2-saturated water with 15 µg/mL phenolred was added to the cuvette to start the reaction at 2 °C. The reaction was monitored spectrophotometrically at 558 nm as decrease of absorbance . The Wilbur-Anderson Unit (WAU) [g at 4 °C for 5 min, and protein content of the supernatant was determined using the Bradford assay [Activity of βCA1 was measured according to ,23. Recoit (WAU) ,24 was drd assay . DefinedEllman’s reagent was used to quantify the free thiols of βCA1 . AbsorptA. thaliana Col_0 and βca1 knock out lines (SALK_106570C and SALK_131447) were obtained from the Salk Institute for Biological Studies [−2 s−1) at 21 °C and 14 h dark at 19 °C with 50% relative humidity. Leaves of 5-week-old plants were cut and placed on a Petri dish on ice in darkness or high light (HL: 800 µmol photons m−2 s−1), respectively. Control leaves were stressed just with HL for 1 h. After indicated time points, samples were taken and immediately frozen in liquid nitrogen. To combine cold stress with HL, the pots were placed in ice-water in HL for 1 h. Afterwards, the plants were harvested and immediately frozen in liquid nitrogen. Studies  and growm value of −366 mV for DTT at pH 8.0 [v/v) glycerol, 4% (w/v) sodiumdodecylsulfate (SDS), 0.01% (w/v) bromophenol blue) and loaded on a non-reductive SDS-PAGE (12%). Afterwards, the gel was blotted to nitrocellulose. Proteins were detected using specific anti-βCA1 antibody, peroxidase-labeled secondary antibody against rabbit, ECL Substrate , and X-ray films.Redox titration was done as previously described by . Ratios t pH 8.0 . After ig for 5 min at room temperature. Protein content of the supernatant was determined according to [Pulverized plant material was vortexed with 5 mM mPEG-Mal in 40 mM Tris-HCl pH 7.2 and incubated for 30 min in dark at room temperature. The samples were centrifuged at 20,000× rding to . Then, 5Pisum sativum βCA (pdb: 1ekj). From the octamer, only a dimeric subunit was used for further analysis with PyMOL version 2.4.0 [βCA1 structure was obtained from SWISS-MODEL  based onon 2.4.0 .https://bibiserv.cebitec.uni-bielefeld.de/concysfind). ConCysFind evaluates the likelihood of functional conservation of post-translational modification-relevant amino acids by employing a comparative phylogeny-based approach featuring 21 plant proteomes. For every species, the most similar protein sequence to the input query containing the Cys serves as basis for the calculation of a Cys-Score, which indicates the degree of conservation of a particular Cys at a relative position over all 21 species. TSV input function and UniProt ID of A. thaliana βCA1 (P27140) were used for submission of the survey. Analysis was performed following ConCysFind-default settings . A particular Cys was considered conserved when featuring a Cys-Score > 0.5 and a corresponding p-value < 0.5.In silico analyses for Cys conservation were performed with the publicly available pipeline ConCysFind  gave longitudinal dimensions of 7 nm for the dimer . Proteins were isolated from the HL-treated plant material and used for CA activity measurement. The differences in CA activity of WT plants in comparison to βca1 knock out plants is remarkable. Since the only difference in the two SALK lines in comparison to the WT plants is the lack of βCA1 protein, the differences in activity reflects the βCA1 activity. βCA1 activity represents about 80% of the total CA activity  . TRX-y1 3− to CO2 and may facilitate the substrate delivery to RubisCO. The contribution of βCA was simulated by mathematical modeling and ranged between 5% and 9% [While these considerations in context of redox regulation appear straight forward, the function of βCA1 in chloroplasts of C3-plants and the significance of thiol switching of βCA1 remain to be explored. βCA1 catalyzes the conversion of HCO% and 9% ,47. Whil% and 9% ).3− on the oxygen evolving complex [3− has a positive effect on photophosphorylation, which is the ATP synthesis of thylakoids. Fedorchuk et al. [3− concentration and its transport into the lumen. Following import, CA-dependent dehydration releases H+ and CO2. The H+ may participate in easing the reduction of dehydroascorbate to ascorbic acid.Interestingly, a certain fraction of chloroplast CA is associated with the thylakoid membrane where it interferes with photosystem II . Inhibit complex . HCO3− hk et al.  suggesteDioscorins, a major tuber storage protein of Yam, functions both as CA and dehydroascorbate reductase (DHAR) . The mai+ could occur in ATP synthesis and the CO2 evades from the thylakoids as mentioned above. Redox regulation is well-established for processes in photosynthetic electron transport, e.g., thiol switching of F-ATP synthase to control photophosphorylation [Another protein usage of the Hrylation  and cyclrylation . TherefoOur study proves the existence of a thiol switch mechanism controlling chloroplast βCA1 activity. The thiol redox state of βCA1 is linked to the TRX system. This result provides an explanation for the detection of βCA1 in earlier redox proteomics analyses. Among the tested recombinant TRXs, TRX-y1 and TRX-y2 most efficiently activated oxidized inactive βCA1. Other chloroplast TRXs and TRX-like proteins like CDSP32 were inefficient or unable to activate βCA1. Mutation of any Cys residue of βCA1 partially or completely inhibited its activity. The thiol redox switch had little influence on conformation and did not affect quaternary structure. Changes in thiol redox state of βCA1 occur in vivo as detected in leaf extracts of wildtype Arabidopsis upon exposure to high light. It may be hypothesized that thiol redox regulation of CAs is needed for fine-tuned adjustment of photosynthetic processes such as photophosphorylation.