Cytophaga hutchinsonii (ChPPK) had the best catalytic activity for the substrates ADP and polyP6. An extracellular–intracellular dual system was constructed to high-throughput screen for better catalytic activity of ChPPK mutants. Finally, the specific activity of ChPPKD82N-K103E mutant was increased by 4.3 times. Therefore, we focused on the production of L-theanine catalyzed by GMAS as a model of ATP regeneration. Supplying 150 mM ATP, GMAS enzyme could produce 16.8 ± 1.3 g/L L-theanine from 100 mM glutamate. When 5 mM ATP and 5 U/mL ChPPKD82N-K103E were added, the yield of L-theanine was 16.6 ± 0.79 g/L with the conversion rate of 95.6 ± 4.5% at 4 h. Subsequently, this system was scaled up to 200 mM and 400 mM glutamate, resulting in the yields of L-theanine for 32.3 ± 1.6 g/L and 62.7 ± 1.1 g/L, with the conversion rate of 92.8 ± 4.6% and 90.1 ± 1.6%, respectively. In addition, we also constructed an efficient ATP regeneration system from glutamate to glutamine, and 13.8 ± 0.2 g/L glutamine was obtained with the conversion rate of 94.4 ± 1.4% in 4 h after adding 6 U/ mL GS enzyme and 5 U/ mL ChPPKD82N-K103E, which further laid the foundation from glutamine to L-theanine catalyzed by GGT enzyme. This proved that giving the reaction an efficient ATP supply driven by the mutant enzyme enhanced the conversion rate of substrate to product and maximized the substrate value. This is a positively combination of high yield, high conversion rate and high economic value of enzyme catalysis. The mutant enzyme will further power the ATP-consuming biocatalytic reaction platform sustainably.ATP, an important cofactor, is involved in many biocatalytic reactions that require energy. Polyphosphate kinases (PPK) can provide energy for ATP-consuming reactions due to their cheap and readily available substrate polyphosphate. We determined the catalytic properties of PPK from different sources and found that PPK from The online version contains supplementary material available at 10.1186/s13068-023-02361-9. Adenosine triphosphate (ATP) is an essential high-energy phosphate compound in living organisms . It provEscherichia coli. PPK2s show the opposite characteristics which can catalyze polyP as a donor to adenosine phosphorylation [At present, many enzymes have been reported to be used for ATP regeneration, such as pyruvate kinase (PK), creatine kinase (CK), acetate kinase (AK) and polyphosphate kinase (PPK) . Howeverrylation . PPK2s crylation . PPK2 I rylation , PPK2 IIrylation  and PPK2rylation .In this work, we performed substrate profiling and substrate tolerance analysis for PPK from different sources and constructed a high-throughput dual system screening with intracellular and extracellular to screen the better enzyme activity of PPK-mutants. Molecular docking and structural analysis revealed the catalytic efficiency of the enzyme could be highly improved by enlarging the dual-substrate channel cavity. At the same time, this mutation could improve the utilization range of the substrate polyphosphates and the tolerance at different concentrations of polyphosphate. Therefore, based on this mutant enzyme, we developed a platform model for ATP regeneration to produce L-theanine and glutamine. With greatly reduced cost and broadened substrate selection range, this efficient ATP regeneration system will be further used in biocatalytic reactions.BlPPK (WP_112868569.1), CgPPK (WP_074495911.1), PaPPK2I (WP_003112634.1), PaPPK2II (WP_070331057.1), PaPPK2III (WP_003091935.1) were amplified from E. coli, C. glutamicum, Pseudomonas aeruginosa. The genes of EbPPK from Erysipelotrichaceae bacterium [ChPPK from Cytophaga hutchinsonii (WP_011583516.1), gmas (MN514852.1) from Methylovorus mays were synthesized by GENEWIZ Bio Inc , respectively. The glnA (NZ_LOQW01000025.1) gene was amplified from the genome of C. glutamicum. All genes were introduced into the plasmid pET-28a ( +) or pXMJ-19. These recombinant plasmids were then transformed to E. coli BL21 (DE3). E. coli was grown in LB liquid medium-containing 5 g/L yeast extract, 10 g/L tryptone, and 10 g/L NaCl at 37 °C. LB solid medium was that 1.8 g agar were added per 100 mL of LB liquid medium. 50 μg/mL kanamycin or 10 μg/mL chloramphenicol were added to the LB medium. PolyP3, polyP6, and polyPn (n > 10) were purchased from Aladdin  Co., LTD. Hexokinase and glucose-6-phosphokinase were purchased from Merck Sigma-Aldrich Co., LTD. L-Glutamate , L-cysteine , L-glycine , and glutathione  were purchased from Sangon Biotechnology  Co., LTD. The random mutation kit was purchased from Beijing TIANDZ Biotechnology Co., LTD., and the other reagents were purchased from Sinopharm Chemical Regent Co., LTD. Prices for all chemicals in the manuscript refer to Macklin .All strains and plasmids used or constructed in this work are listed in Additional file acterium   and glutamine synthetase (GS) overexpression was induced by adding 0.2 mM isopropyl-β-d-thiogalactoside (IPTG) until the for 18 h . The celeviously .2, 10 mM ADP, 50 mM polyP6, 5 U HK, 5 U G6PD and enzyme. The reaction was initiated by the addition of polyP6. To verify the effects of polyP-chain length, we select polyP3, polyP6, polyPn (n > 6) in reaction mixtures. 50 mM, 100 mM, 150 mM and 200 mM polyP6 were applied for exploring the effects of polyP6 concentration. The mixture containing 10 mM ADP or 10 mM AMP were also analyzed. The reaction was incubated at 35℃ for 30 min and the mixture was measured at 340 nm for absorbance value. One unit of PPK (1 U) is defined as the amount of enzyme required to produce 1 μmol ATP per minute.Hexokinase (HK) is responsible for catalyzing the first step in glycolysis. It causes glucose and ATP to form glucose-6-phosphate and ADP. Glucose-6-phosphate dehydrogenase (G6PD) is a key enzyme in the pentose phosphate pathway and can convert glucose-6-phosphate and NADP to NADPH and gluconolactone 6-phosphate. The enzKm and Vmax values of the enzyme were determined by double reciprocal method [Km and Vmax values of the WT, D82N and K103E were measured of 100 mM Tris–HCl (pH 8.0) with different concentrations of ADP (0.1 to 5 mM) and polyP6 (1 to 7.5 mM), respectively. The kinetic parameters of ADP and polyP6 were determined by the enzyme activity method described above. All experiments were performed in triplicate.The l method . The Km Enzyme activity of GS was determined as described previously . The reachppk gene was performed by TIANDZ error-prone PCR kit (https://tiandz.etlong.com/). The plasmid pXMJ-19-tac-ChPPK was used as template, and the F and R primers are seen Additional file ChPPK from error-prone PCR, accompanied by the changes of MgCl2 and MnCl2 [rrnB P1-GFP and then transformed to the E. coli BL21 (DE3). The transformed strains were spread on LB-agar plates containing 10 μg/mL chloramphenicol and incubated at 37 ℃ for 24 h for obtaining transformants (BL21/pXMJ-19-tac-ChPPKmuts- rrnB P1-GFP).The error-prone PCR of nd MnCl2 . The fra2O2 under weak acidic conditions [ChPPKmuts-rrnB P1-GFP) were cultured in 24-well plates containing 3 mL LB medium for 12 h at 37 ℃, and then 1% of the inoculum were transferred to the new 24-well plate that cultured at 37℃ for 2 h. 0.5 mM IPTG were added to the 24-well plate for induction at 16 ℃ for 12 h. The culture mediums were washed twice with PBS buffer (pH 7.4), and then the cells were collected for subsequent experiments. The mutants with high enzyme activity were screened by TMB-H2O2-ATP chromogenic method [6, 5 mM MgCl2, 10 mM ADP and the collected cells. The solutions were reacted at 35 ℃ for 1 h. After centrifugation at 15,984 g for 1 min, 400 uL of the supernatant was taken and added to the solution B of 200 mM PBS buffer (pH 5.0) containing 5 mM TMB, 100 mM H2O2 at 40 ℃ for 30 min. The reaction solutions were terminated with 10% trichloroacetic acid. The solutions were screened for mutants with improved enzyme activity by detecting the absorbance value at 652 nm. In order to make the reaction more sensitive to the detection of ATP, we first optimized the concentration of TMB and H2O2. We observed the effects of different concentrations of TMB  and H2O2  on the absorbance values of different concentrations of ATP .The extracellular high-throughput screening method is based on the chromogenic reaction. It was reported that 3,3′,5,5′-tetramethylbenzidine (TMB) has a benzidine structure, which contains two amidogens that are easy to oxidize. ATP has the property of peroxidase, which can catalyze the oxidation of TMB by Hnditions . The oxinditions . The trac method . First, rrnB P1 has been shown to depend on ATP concentration in E. coli cells [rrnB P1 form a very short active complex that requires high concentrations of ATP to initiate transcription of rRNA. We used the strains BL21/pXMJ-19-tac-ChPPKmuts-rrnB P1-GFP to detect the fluorescence value. The mutants were selected and cultured in 24-well plates at 37 ℃ for 12 h. 1% of the inoculum were transferred to the new 24-well plate that cultured at 37 ℃ for 2 h. 0.5 mM IPTG, 5 mM ADP and 8 mM polyP6 were added to the 24-well plate for induction at 16 ℃ for 12 h. The strains were collected and were suspended in PBS buffer. The fluorescence value was detected by Elisa, with the OD600 value. We selected mutants with high fluorescence value/OD600 value for subsequent experiments, compared with the wild type.The activity of the ribosomal RNA promoter li cells . RNA polChPPK were mutated to the remaining 20 amino acids by site-directed PCR [chppk was used as the DNA template. The plasmids were transformed into E. coli BL21 (DE3) and spread on LB-agar plates containing 10 μg/mL chloramphenicol and incubated at 37 ℃ for 24 h. The mutants with correct DNA sequencing were selected for enzyme activity analysis.Residues D82 and K103 in wild-type cted PCR , respectChPPK was 6AN9. Docking between substrate and protein was completed by Schrödinger-glide dock and the best docking position was selected by docking analysis. Import the docking box and ligand, set the docking accuracy to SP (standard precision). The root mean square deviation (RMSD) reflects the position change degree of the molecular structure with time (shift degree), while the root mean square deviation (RMSF) represents the freedom degree of movement of each atom in the molecule [The PDB ID of the protein molecule . RMSD anmolecule . MD simu6, 20 g/L ADP, 10 g/L MgCl2 and the pure enzymes (5 U/mL). The reaction was at 37 ℃, 150 r/min in the shaker for 24 h. Samples were placed in boiling water for 5 min to terminate the reactions and measured the production of ATP.Systems for the ATP-producing capacity of PPK enzymes (200 mL) included Tris–HCl , 20 g/L polyP4)2SO4, 50 mM polyP6, 5 mM ATP, 50 mM MgCl2, 5 U/mL PPK, 10 U/mL GMAS. The reaction was at 37 ℃, 150 r/min in the shaker for 24 h. System of glutamine production (200 mL) by GS enzyme consists of Tris–HCl , 100 mM L-glutamate, 100 mM (NH4)2SO4, 50 mM polyP6, 5 mM ATP, 50 mM MgCl2, 5 U/mL PPK, 6 U/mL GS. The reaction was at 37 ℃, 150 r/min in the shaker for 24 h. The samples were placed in boiling water for 5 min to terminate the reactions. Samples were centrifuged at 15,984 g for 5 min and measured the yield by HPLC.System of L-theanine production (200 mL) by GMAS enzyme consists of Tris–HCl , 100/200/400 mM L-glutamate, 100/200/400 mM ethylamine, 200 mM  , 4. The Escherichia coli (BlPPK), Corynebacterium glutamicum (CgPPK), Pseudomonas aeruginosa , Erysipelotrichaceae bacterium (EbPPK), Cytophaga hutchinsonii (ChPPK) were selected and all genes were introduced into the plasmid pET-28a. The plasmids have been successfully expressed in E. coli BL21(DE3). SDS-PAGE analysis indicated that the five PPKs were expressed successfully . Therefore, EBPPK and ChPPK were chose to express on the pXMJ-19 plasmids. The results showed that the two proteins were expressed clearly and purified successfully  may block the entrance of ADP binding pocket for the lower enzyme activities. Due to the sensitivity of PPK to phosphate-chain length [6 at different concentrations on the enzyme activity were proposed. The ability of ATP formation was gradually inhibited with the increase of polyP6 concentration .In order to further improve the enzyme activity of 2O2-ATP chromogenic method  is related to intracellular ATP content in E. coli and that RNA polymerase holoenzyme and rrnB P1 form a very short active complex that requires ATP to initiate transcription of rRNA. We constructed the plasmid pXMJ-19-tac-ChPPKmuts-rrnB P1-GFP to verify whether the fluorescence intensity of GFP protein under the rrnB P1 promoter was responsive to the concentration of ATP  was constructed to verify the effect of different ATP concentrations (0–10 mM) on fluorescence values. With the increase of ATP concentration, the fluorescence value/OD600 value gradually increased . In particular, the enzyme activity of the eight mutants increased significantly  obtained the highest enzyme activity with the 93.3% improvement in the saturated mutation library of D82, while D2 (D82H) and D3 (D82T) also improved the 21.5% and 27.0% of enzyme activity compared the wild mutant  and the molecules of polyP6 and ADP were docked by Schrodinger. The ChPPK protein is composed of a structure like a "sandwich" , while ADP forms interaction of ions with residues K81, D82, K103 and R133    ADP into 44±0.4 mM (18.8±0.19 g/L) ATP and the time-space yield of ATP was 4.7±0.05 g/L/h and exhibited the best ability of ATP production compared to the other six enzymes  at 4 h under the combined action of GMAS and ChPPKD82N-K103E. Compared to the wild enzyme ChPPK, the yield of the L-theanine improved 53.7%. ChPPKD82N-K103E was responsible for the similar yield of L-theanine to that produced by adding sufficient ATP in the substrates. Subsequently, the reactions were scaled to 200 mM glutamate in 200-mL reaction system, respectively. ChPPKD82N-K103E still revealed the similar ATP supply capacity. The yield of L-theanine was reached to 32.3±1.6 g/L with 92.8±4.6 % substrates converted within 6 h. Finally, we expanded the substrate concentration to 400 mM to produce L-theanine, and the results are shown in Fig. ChPPKD82N-K103E, the addition of 5 mM ATP was used to produce L-theanine. Under the ATP regeneration system of ChPPKD82N-K103E, the yield of L-theanine reached 62.7±1.1 g/L with a conversion rate of 90.1%. In summary, the mutant enzyme was shown to replace the addition of the substrate ATP efficiently and similarly, greatly saving the reaction cost. The final yield of L-theanine could reach 62.7±1.1 g/L, with a high conversion rate of 90.1±1.6 %. As we know, the high toxicity of ethylamine requires that the residual amount of this substrate should be reduced as much as possible in the reaction. We tested the residual amount of ethylamine in the final reaction solution and found that the yields of ethylamine were still low as expected (0.29 g/L to 3.5 g/L)  reaction was carried out to obtain 94.4±1.44 mM glutamine in 4 h . In the one-step synthesis of L-theanine catalyzed by GMAS enzyme, we enhanced the recycling and regeneration of ATP by mutation, improved the conversion capacity of glutamate substrate, and finally obtained 62.7±1.1 g/L L-theanine in 200 mL system. At the same time, we also tried to synthesize glutamine from glutamate to reduce the cost of L-theanine production catalyzed by GGT enzyme. Finally, we obtained 13.8±0.2 g/L glutamine with the conservation rate of 94.4±1.4 %. Due to the difference in the optimal pH between GS and GGT, we will further target the enzymatic modification of GGT to drop the pH of this enzyme and better cascade the production of L-theanine.GGT was considered as the other pathway to catalyze the production of L-theanine Fig. a. AlthouChPPKD82N-K103E was increased to 430.2% and was considered as the highest catalytic efficiency among the major polyphosphate kinases. In the cascade catalysis or in vitro biosynthesis system, in addition to preferentially selecting ATP with low energy consumption or avoiding the use of ATP, an ATP regeneration system with low cost, high activity and high yield is also one of the strategies of synthetic biology. This efficient ATP regeneration system can not only produce glutamine from glutamate to further produce L-theanine, but also directly produce L-theanine from glutamate in one step. Adding GMAS and ChPPKD82N-K103E, L-theanine was produced by 62.7 ± 1.1 g/L with high conversation rate and low cost. To the best of our knowledge, this is a positively combination of high yield, high conversion rate and high economic value of enzyme catalysis. After achieving the best efficient PPK-mutant enzyme, the addition of ChPPKD82N-K103E can significantly improve the efficiency and sustainability of ATP regeneration, which will enhance the catalytic efficiency and save the cost in energy-demanding catalytic reactions.In this research, we displayed a high-throughput dual system screening with intracellular and extracellular. With the improvement of substrate spectrum and tolerance, the catalytic activity of ChPPKD82N-K103E in E. coli and are currently optimizing this system for whole-cell catalytic L-theanine production as the simple form of catalysis. The sustainability of efficient ATP-regeneration demonstrated by ChPPKD82N-K103E will be further used in biocatalytic reactions to produce high-value food chemicals such as glutathione, SAM and so on. It will further be a biological reaction platform to strengthen the sustainable supply of ATP-regeneration in enzyme expression intensity, enzyme immobilization and other strategies.In addition, we have constructed a co-expression system for GMAS-Additional file 1:Fig. S1. An evolutionary tree of PPK enzymes. Fig. S2. a Crude enzyme expression of PPK. Lane 1: marker; lane 2: BL21/28a; lane 3: BL21/28a-PaPPKI; lane 4: BL21/28a-PaPPKII; lane 5: BL21/28a-PaPPKIII; lane 6: BL21/28a-BlPPK; lane 7: BL21/28a-CgPPK; lane 8: BL21/28a-EbPPK; lane 9: BL21/28a-ChPPK; lane 10: marker; lane 11: BL21/pXMJ19; lane 12: BL21/pXMJ19-EbPPK; lane 13: BL21/pXMJ19-ChPPK. . b Pure enzyme expression of PPK. Lane 1: marker; lane 2-4: PaPPKI; lane 5-6: PaPPKII; lane 7: PaPPKIII; lane 8: BlPPK; lane 9: EbPPK; lane 10: ChPPK; lane 11: CgPPK. Fig. S3a Absorbance values at different concentrations of TMB. b Absorbance values at different concentrations of H2O2. c Color diagram of TMB-ATP-H2O2. Table S1. Biocatalytic reactions involving ATP to produce high-value chemicalsa. Table S2. The number of bases of the PPK protein. Table S3. The capacity and yield of PPK enzymes to produce ATP. Table S4. The ATP regeneration system to produce L-theanine and glutamine. Table S5. Strains and plasmids used in this work. Table S6. Primers used in this work.


The TE of SLC22A11 for IS was similar to pCS. For OAT1, however, IS is an excellent substrate. With OAT1 and p-aminohippuric acid, our study revealed an influence of transporter abundance on the outcomes of the hyperosmolarity assay; very high transport activity confounded results. SLC22A11 was found to insert both pCS and IS into the plasma membrane, whereas OAT1 conveys these toxins to the cytosol. These disparate transport mechanisms bear profound ramifications for toxicity. Membrane insertion might promote membrane damage and microvesicle release. Our results underscore the imperative for detailed structural inquiries into the translocation of small molecules.Chronic kidney disease (CKD) is a global health concern affecting millions worldwide. One of the critical challenges in CKD is the accumulation of uremic toxins such as p-cresol sulfate (pCS) and indoxyl sulfate (IS), which contribute to systemic damage and CKD progression. Understanding the transport mechanisms of these prominent toxins is essential for developing effective treatments. Here, we investigated whether pCS and IS are routed to the plasma membrane or to the cytosol by two key transporters, SLC22A11 and OAT1. To distinguish between cytosolic transport and plasma membrane insertion, we used a hyperosmolarity assay in which the accumulation of substrates into HEK-293 cells in isotonic and hypertonic buffers was measured in parallel using LC-MS/MS. Judging from the efficiency of transport (TE), pCS is a relevant substrate of SLC22A11 at 7.8 ± 1.4 µL min With diminished kidney function, metabolic waste products that are excreted by the kidney under normal conditions accumulate in the body and cause systemic damage. The increased retention of so-called uremic toxins further deteriorates renal function, contributing to the development of chronic kidney disease (CKD) ,2. CKD iSLC22A6) and OAT3 (SLC22A8) are found in the basolateral membrane of proximal tubular cells; their substrate spectrum, besides pCS and IS, encompasses many other anionic compounds, including metabolites, toxins, and drugs [pCS and IS are normally excreted from the human body through the renal proximal tubule. The two toxins are mainly cleared from the blood compartment via uptake catalyzed by organic anion transporter 1 (OAT1) and OAT3 ,13,14. Ond drugs . In addind drugs ,16,17. TEscherichia coli, can insert certain amphipathic or predominantly hydrophobic molecules into the membrane through a lateral opening [We have previously uncovered that SLC22A11 transports the substrates E3S and UA via fundamentally different mechanisms . Our dat opening . In addi opening . SurprisTo better understand the pathophysiological mechanisms of toxicity in humans caused by the uremic toxins pCS and IS and their roles in CKD progression, it is important to identify the underlying transport mechanisms in detail. The current belief invariably holds that uremic toxins such as pCS and IS are transported by organic anion transporters into and out of the cytosol. However, our recent results might challenge this assumption: due to the structural similarity to E3S see , transpoWe have recently established a hyperosmolarity assay to distinguish between regular transport of small molecules into the cytosol and insertion into the plasma membrane . In thisThe primary aim of the present study was to investigate the mechanisms of transport of pCS and IS via human OAT1 and SLC22A11 in 293 cells using the hyperosmolarity assay. Our results indicate that OAT1 transports pCS and IS into the cytosol, whereas SLC22A11 transports pCS and IS into the plasma membrane.The mechanisms of transport of the uremic toxins IS and pCS by SLC22A11 and OAT1 were investigated through hyperosmolarity experiments developed previously . Transpo−1 to a maximum of 3.8 ± 0.1 nmol mg protein−1 at 400 mM mannitol (−1 mg protein−1 (unpublished). We therefore repeated the assay with reduced expression of OAT1. The reduction was achieved by titrating doxycycline in the culture medium , a model substrate for OAT1, was examined. Surprisingly, OAT1-expressing cells showed a slight increase in accumulation under hyperosmolar conditions after incubation with 10 µM pAH for 20 min, from 3.2 ± 0.1 nmol mg proteinmannitol A. This wmannitol , insertie medium . At 3 ng−1 to a minimum of 19 ± 1 pmol mg protein−1 at 800 mM mannitol  and 0.58 ± 0.01 nmol mg protein−1 (400 mM mannitol). In contrast, the accumulation of IS by OAT1 over time was inhibited by hyperosmolarity; after 60 min, the accumulation of IS was 4.6 ± 0.1 nmol mg protein−1 (control) and 3.3 ± 0.1 nmol mg protein−1 (400 mM mannitol)  were incubated with 10 μM IS in uptake buffer with or without 400 mM mannitol A. The acannitol) A. This i−1 (0 mM mannitol) to a maximum of 0.45 ± 0.03 nmol mg protein−1 at about 400 mM mannitol (−1 (0 mM mannitol) to a minimum of 2.0 ± 0.1 nmol mg protein−1 at about 600 mM mannitol. In experiments with expression reduced to approximately 10% uptake , IS levels in OAT1-expressing cells actually decreased by more than half, from 0.68 ± 0.01 nmol mg protein−1 (0 mM mannitol) to 0.28 ± 0.01 nmol mg protein−1 (800 mM mannitol) (−1 (0 mM mannitol) to a maximum of 0.67 ± 0.01 nmol mg protein−1 at about 600 mM mannitol (In long-term incubations (60 min) in which the uptake of IS into SLC22A11-expressing cells (high expression) was examined as a function of mannitol concentration, the IS levels increased from 0.20 ± 0.01 nmol mg proteinmannitol B. In conannitol) C. In SLCmannitol C. These −1 (control) and 0.37 ± 0.01 nmol mg protein−1 (400 mM mannitol) (−1 (control) and 0.61 ± 0.01 nmol mg protein−1 (400 mM mannitol) (In time course experiments (high expression), the accumulation of pCS by SLC22A11 over time was stimulated by hyperosmolarity; after 20 min, the accumulation of pCS was 0.26 ± 0.01 nmol mg proteinannitol) A. In conannitol) . These r−1 (0 mM mannitol) to 0.46 ± 0.01 nmol mg protein−1 (800 mM mannitol) (−1 (0 mM mannitol) to 1.4 ± 0.1 nmol mg protein−1 (800 mM mannitol). In experiments with reduced transporter expression, pCS levels in OAT1-expressing cells decreased from 0.48 ± 0.01 nmol mg protein−1 (0 mM mannitol) to 0.22 ± 0.01 nmol mg protein−1 (800 mM mannitol) (−1 (0 mM mannitol) to 0.20 ± 0.01 nmol mg protein−1 (800 mM mannitol) (In incubations in which the uptake of pCS into SLC22A11-expressing cells (high expression) was examined at equilibrium as a function of mannitol concentration, pCS concentrations increased from 0.26 ± 0.01 nmol mg proteinannitol) A. In OATannitol) C. In conannitol) C. These −1 (control) and 0.077 ± 0.003 nmol mg protein−1 (mannitol). This suggests that IP, like IS and pCS, is inserted into the membrane by SLC22A11. No uptake of IC or IAA into 293 cells via SLC22A11 was observed (To investigate whether the sulfate residue in indoxyl sulfate (IS) can be substituted by other moieties with a negative charge in the membrane insertion by SLC22A11, the transport of the uremic toxin indole-3-acetic acid (IAA) as well as indole-3-carboxylic acid (IC) and indoxyl phosphate (IP)  was examobserved . In this study, we examined whether the prominent uremic toxins pCS and IS are actually inserted into the plasma membrane by SLC22A11 or OAT1. This hypothesis was based on the structural similarity of pCS and IS to the steroid hormone precursor E3S ; these cResults for pCS and IS are consistent. Most importantly, the transport of both uremic toxins by OAT1 and SLC22A11 was fundamentally different. With SLC22A11, the uptake curves of pCS and IS B resemblTransport to these separate compartments likely results in different mechanisms of toxicity. When pCS and IS enter the cytosol of tubule cells, for example, they can bind to and activate the human aryl hydrocarbon receptor (AHR) ,28, indunot decreased by the addition of mannitol, but even slightly increased  was higher compared to that of IS (0.23 µL min−1 mg protein−1) or E3S . This cin values for expression on and off), was 7.8 ± 1.4 µL min−1 mg protein−1. Thus, pCS is a relevant substrate of SLC22A11 but not as good as E3S (40 ± 6 µL min−1 mg protein−1 [−1 mg protein−1). The efficiency of transport of IS by SLC22A11 was similar to that of pCS at 5.6 ± 0.3 µL min−1 mg protein−1 ; OAT1—wrotein−1 —translocrotein−1 A. For OAe of pCS .alias OAT4 was demonstrated in transfected cells [Our data agree with a previous report where uptake and efflux of IS by human SLC22A11 ed cells ; howeverTo investigate whether the sulfate residue in indoxyl sulfate (IS) can be substituted by other moieties with a negative charge in the membrane insertion by SLC22A11, the transport of the indole derivatives IAA, IC, and IP was examined using the hyperosmolarity assay. For IP, the uptake into 293 cells by SLC22A11 was stimulated by hyperosmolarity after 60 min of incubation , indicatalias EMT; gene symbol SLC22A3), a lateral access site (the V-site) was identified through which OCT3 could transport its hydrophobic substrates out of or into the plasma membrane [Transporter-mediated membrane insertion of suitable compounds is apparently not limited to anionic solutes. Based on the cryo-EM structure of the organic cation transporter 3  included 4-aminohippuric acid (08088), estrone-3-sulfate sodium salt (E0251), indole-3-acetic acid , indole-3-carboxylic acid (284734), indoxyl phosphate disodium salt (I5505), indoxyl sulfate potassium salt (I3875), potassium 4-methylphenyl sulfate , and uric acid sodium salt (U2875). All other chemicals were at least of analytical grade. Stock solutions, each at 10 mM, were prepared by dissolving estrone-3-sulfate (E3S) in methanol; indole-3-carboxylic acid (IC) in ethanol; 4-aminohippuric acid (pAH), indoxyl phosphate (IP), indoxyl sulfate (IS), and p-cresol sulfate (pCS) in water; and indole-3-acetic acid (IAA) and uric acid (UA) in water with 0.1 M sodium hydroxide.The transporter cDNAs used in this study were of human origin. The SLC22A11 cDNA was expressed from pEBTetLNC , and OAT2 atmosphere. The growth medium was Dulbecco’s Modified Eagle Medium  supplemented with 10% fetal bovine serum , 100 U mL−1 penicillin, and 0.1 mg mL−1 streptomycin ; in some experiments with SLC22A11, 10 μg mL−1 ciprofloxacin was used instead of penicillin/streptomycin. The medium was changed every 2–3 days, and the culture was split every 5 days. Stably transfected cell lines were generated, as reported previously [−1 puromycin  to maintain plasmids for up to 8 weeks in culture. Nevertheless, only cells whose transfection was not older than 5 weeks were used for the experiments.A total of 293 cells , a transformed cell line derived from human embryonic kidney, were grown as adherent culture in plastic culture flasks  at 37 °C in a humidified 5% COeviously , using Teviously  are prop−1 poly-L-ornithine  in 0.15 M boric acid-NaOH pH 8.4 and grown to a confluence of at least 70%. To turn protein expression on, cells were cultivated for at least 20 h with 1 μg mL−1 doxycycline  in growth medium unless otherwise indicated. Uptake buffer contained 125 mM NaCl, 25 mM HEPES-NaOH pH 7.4, 5.6 mM (+) glucose, 4.8 mM KCl, 1.2 mM KH2PO4, 1.2 mM CaCl2, and 1.2 mM MgSO4.For measurement of solute uptake, cells were seeded in 6 cm diameter polystyrol dishes  precoated with 0.1 g lv/v Triton X-100 in 50 mM TRIS-HCl pH 7.4 was used as the lysis buffer. Protein was measured using the BCA (bicinchoninic acid) assay  with bovine serum albumin as the standard.Incubations were performed in a water bath at 37 °C for 10 min or longer. Cells were washed at least twice with 4 mL of uptake buffer at 37 °C, and afterwards, incubations were started by adding 2 mL of 10 μM substrate in uptake buffer. After incubation, cells were washed three times with 4 mL of ice-cold uptake buffer, lysed for at least 20 min with 1 mL of methanol, and then stored at −20 °C. In some experiments, the buffer used for washing and uptake was modified as indicated with mannitol . The protein content of MS samples was estimated from 3 paired dishes; here, 0.1% g, 20 °C) of thawed cell lysates, samples were transferred to glass vials, and then 10 μL (20 μL for pAH) of sample was analyzed via HPLC coupled to a triple-quadrupole mass spectrometer. The following system was used: an LC-20AD Prominence HPLC  with a flow rate of 0.2 to 0.4 mL/min coupled to a 4000 Q TRAP  mass spectrometer. A blank or reference sample, which was methanol (HPLC gradient grade), was measured before and between sample measurements. The following HPLC methods were used. E3S, XBridge Shield RP18 column ; A: 10 mM ammonium acetate pH 8.9, B: methanol; 0.2 mL/min gradient flow: 70% B at 0 min, 70% B at 0.5 min, 20% B at 3 min, 20% B at 4 min, 70% B at 5 min, 70% B at 7 min. IAA and IS, Atlantis dC18 column ; A: 0.1% formic acid, B: acetonitrile with 0.1% formic acid; 0.3 mL/min gradient flow: 10% B at 0 min, 10% B at 0.25 min, 80% B at 3 min, 80% B at 5.5 min, 10% B at 7 min, 10% B at 10 min. IC and IP, Atlantis dC18 column; A: 0.1% formic acid, B: methanol with 0.1% formic acid; 0.25 mL/min gradient flow: 10% B at 0 min, 10% B at 0.25 min, 90% B at 3 min, 90% B at 6 min, 10% B at 7 min, 10% B at 10 min. pAH, Atlantis HILIC Silica column  A: 10 mM ammonium acetate pH 4.0, B: methanol with 0.1% formic acid; 0.4 mL/min gradient flow: 80% B at 0 min, 80% B at 0.25 min, 20% B at 2 min, 20% B at 3 min, 80% B at 4 min, 80% B at 5 min. pCS, Atlantis dC18 column; A: 0.1% formic acid, B: methanol with 0.1% formic acid; 0.3 mL/min gradient flow: 10% B at 0 min, 10% B at 0.25 min, 80% B at 3 min, 80% B at 5.5 min, 10% B at 7 min, 10% B at 13 min. UA, iHILIC-(P) Classic column ; A: 10 mM ammonium acetate pH 8.9, B: methanol; 0.2 mL/min gradient flow: 80% B at 0 min, 80% B at 0.25 min, 20% B at 4 min, 20% B at 5 min, 80% B at 9 min, 80% B at 10 min. We used atmospheric pressure ionization with positive or negative electrospray. For quantification (scan time 150 ms), the optimal collision energy for nitrogen-induced fragmentation in the second quadrupole was determined for each analyte. From the product ion spectra, the following fragmentations were chosen for selected reaction monitoring ): E3S, 349, 269, N42; IAA, 174, 130, N14; IC, 160, 116, N24; IP, 212, 79, N22; IS, 212, 80, N30; pAH, 195, 120, P15; pCS, 187, 107, N30; and UA, 167, 124, N22. For each analyte, the area of the intensity vs. time peak was integrated. Linear calibration curves were constructed (weighting 1/y2) from at least six standards, which were prepared using control cell lysates as solvent. Sample analyte content was calculated from the analyte peak area and the slope of the calibration curve.After centrifugation  with Equation (1), where cout represents the substrate concentration and kin and kout are rate constants.Results are presented, if not indicated otherwise, as the arithmetic mean ± standard error of the mean (SEM) with In osmolarity experiments, we either used the 3rd-order polynomial function of GraphPad Prism or simple linear regression. In doxycycline titration experiments, we used the sigmoidal, 4PL (four-parameter logistic) curve with Equation (2).t-tests were performed to determine significance; two-tailed p-values are shown in GraphPad style .For


The aim of the present study was to gain a better understanding of the role of brain-derived neurotrophic factor (BDNF) and dopamine D3 receptors in the effects of chronic methamphetamine (METH) on prepulse inhibition (PPI), an endophenotype of psychosis. We compared the effect of a three-week adolescent METH treatment protocol on the regulation of PPI in wildtype mice, BDNF heterozygous mice (HET), D3 receptor knockout mice (D3KO), and double-mutant mice (DM) with both BDNF heterozygosity and D3 receptor knockout. Chronic METH induced disruption of PPI regulation in male mice with BDNF haploinsufficiency (HET and DM), independent of D3 receptor knockout. Specifically, these mice showed reduced baseline PPI, as well as attenuated disruption of PPI induced by acute treatment with the dopamine receptor agonist, apomorphine (APO), or the glutamate NMDA receptor antagonist, MK-801. In contrast, there were no effects of BDNF heterozygosity or D3 knockout on PPI regulation in female mice. Chronic METH pretreatment induced the expected locomotor hyperactivity sensitisation, where female HET and DM mice also showed endogenous sensitisation. Differential sex-specific effects of genotype and METH pretreatment were observed on dopamine receptor and dopamine transporter gene expression in the striatum and frontal cortex. Taken together, these results show a significant involvement of BDNF in the long-term effects of METH on PPI, particularly in male mice, but these effects appear independent of D3 receptors. The role of this receptor in psychosis endophenotypes therefore remains unclear. Methamphetamine  is a widely abused psychostimulant drug that can induce significant and long-lasting neuroplastic and neurotoxic effects on the mammalian brain ,2 and maBrain-derived neurotrophic factor (BDNF) is an abundant growth factor involved in neuroplasticity and development of the nervous system ,10. DefiBDNF has particularly been linked to the expression of the D3 receptor . AlthougAnimal model studies have aimed to elucidate how BDNF promotes neuronal adaptations in target postsynaptic neurons via modulation of D3 receptors ,36. D3 rThe aim of the present study was to gain a better understanding of the role of BDNF and D3 receptors in the effects of chronic METH on endophenotypes of psychosis. We used a chronic, three-week adolescent METH treatment protocol in BDNF HET mice ,37 and iIn this study, we focused on prepulse inhibition (PPI), a model of sensorimotor gating associated with psychosis ,40,41. WStep 1:Crossing of BDNF HET with D3 knockout mice, generating 50% BDNF HET/D3 HET and 50% BDNF wildtype/D3 HET mice with identical genetic background. These animals were used as founding breeders of the double-mutant line.Step 2:Mating BDNF HET/D3 receptor HET mice with BDNF wildtype/D3 receptor HET, generating eight possible genetic combinations of which four were used in the present study : WT, BDNMale and female mice were bred and housed at the La Trobe Animal Research and Teaching Facility . These included wildtype controls (WT), BDNF HET, D3KO, and double-mutant mice (DM) with both D3KO and BDNF haploinsufficiency. BDNF HET mice were originally established through a targeted deletion of the BDNF gene . BecauseMice were housed in individually ventilated cages  in same-sex groups of 2–5 and were provided with a stage and a cardboard tunnel as standard enrichment material. To account for possible litter effects, the number of mice per litter per adolescent treatment did not exceed two per experimental group. No attempts were made to determine the oestrus cycle stage of the female mice as this would have possibly added a level of stress to the protocol. Therefore, it was assumed that the oestrus cycle stage was random during treatment and behavioural testing. Mice had ad libitum access to standard rodent chow and drinking water and were maintained on a 12-h light/dark cycle, 8 am on, 8 pm off. Cages were cleaned every fortnight and all experiments were conducted during the light period. BDNF HET, D3KO, double-mutant, and WT mice were genotyped by Transnetyx .All experiments were approved by the La Trobe University Animal Ethics Committee and were conducted in accordance with the Australian Code of Practice for the Care and Use of Animals enacted by the National Health and Medical Research Council of Australia (NHMRC).During adolescence at 6 ± 0.5 weeks of age, mice were randomly assigned to receive METH ((±)-Methamphetamine-HCl, National Measurement Institute, Pymble, NSW, Australia), or a volume-matched saline vehicle solution (0.9% sodium chloride) to act as control. METH and saline solutions were administered via intraperitoneal (IP) injection at a volume of 5 mL/kg. Mice were administered 1 mg/kg METH Mon–Fri in the first week, 2× daily 2 mg/kg METH in the second, and 2× daily 4 mg/kg in the third week  17,37,5,52,53. Sp < 0.001, ηp2 = 0.881), and male mice were heavier and gained more weight than female mice  = 284.9, p < 0.001, ηp2 = 0.617; sex × time interaction, F = 21.94, p < 0.001, ηp2 = 0.110). BDNF HET and double-mutant mice had higher body weights and greater body weight gain than wildtype and D3 knockout mice  = 77.27, p < 0.001, ηp2 = 0.304; BDNF genotype × time interaction, F = 75.30, p < 0.001, ηp2 = 0.298). There were minor effects of METH pretreatment on body weight gain dependent on the BDNF genotype . A protocol of 104 trials was used over 40–45 min per session. Every session began with a 3-min 65 decibel (dB) background noise phase, followed by eight 115 dB pulse-alone startle stimuli. This block of pulse-alone stimuli was repeated at the end of the PPI session. Within each session, 16 additional pulse-alone trials were pseudorandomly inserted throughout the experiment and startle responses for each mouse were calculated over four blocks of these pulse-alone stimuli to establish habituation (not shown here). The analysis of PPI was conducted on the average of the 32 pulse-alone trials.A prepulse trial was defined as a prepulse stimulus (PP) of 2, 4, 8, or 16 dB above background that preceded the 115 dB pulse at an interstimulus interval (ISI) of 30 ms or 100 ms prior to the startle noise. In total, eight trials were pseudorandomly conducted for each PP intensity at both ISIs. A further eight no-stimulus trials were pseudorandomised within each session to determine any nonspecific motor effects or background vibration. PPI was low and more variable at the 30 ms ISI than the 100 ms ISI and generally did not differ across sex or pretreatment groups; therefore, data presentation focuses on the more commonly used 100 ms ISI.Startle responses were measured through vibrations detected by the PPI chamber and relayed to SR_Lab software  for analysis. PPI is a percentage value calculated by the difference between responses to the eight pulse-alone startle stimuli compared to responses to the pulses preceded by one of the four prepulses.Four PPI sessions were conducted in each mouse with a 2–3 day washout between experiments: (1) A pretest with no injection to acclimatize the mice to the PPI chamber; (2) a saline IP injection directly prior to the PPI session; (3) 3 mg/kg APO (R-(−)-Apomorphine, Sigma Aldrich, Castle Hill, NSW, Australia) injected IP directly prior to the PPI session; (4) 0.2 mg/kg MK-801 ((+)-MK-801 hydrogen maleate, Sigma Aldrich, Castle Hill, NSW, Australia) injected IP 15 min prior to the PPI session. These drugs were selected due to their capacity to disrupt PPI via neurochemical pathways relevant to schizophrenia, particularly dopamine (APO) and NMDA receptor hypofunction (MK-801) . The ordFive to seven days after the last PPI session, mice were tested for baseline and METH-induced locomotor hyperactivity using automated locomotor photocells . For each 180 min session, mice were placed individually into these photocell chambers and allowed to habituate for 60 min. Between the 55 and 60 min time point, each mouse received an acute IP injection before being returned to the photocell where activity was further recorded over the following 120 min. Mice received saline vehicle in the first session, 1 mg/kg METH in the second session, and 3 mg/kg METH in the final session. Three to four days was left between sessions to allow drug washout. Photocell chambers recorded locomotion via the interruption of photoelectric beams positioned along the walls of the chamber. Mouse activity was recorded and analysed by the Activity Monitor software , and outputs for the statistical analysis included cumulative horizontal distance moved every 5 min.2 inhalation and decapitation, and the frontal cortex and striatum were rapidly dissected on a cold plate. Samples were frozen on dry ice and stored at −80 °C until the PCR assay of dopamine D1, D2, and D3 receptors and DAT gene expression.At least one week after the last behavioural test, the mice were euthanised by COTotal RNA was isolated from dissected tissue using Trizol  and single-stranded complementary DNA was synthesized using the QuantiTect Reverse transcription kit . Quantitative PCR was performed with an SYBR Green reagent  using the Rotor-Gene 6000 real-time PCR system . The primers used were:beta actin: F: 5′-GATCATTGCTCCTCCTGAGC-3′, R: 5′-AGTCCGCCTAGAAGCACTTG-3′D1 receptor: F: 5′-CCAGATCGGGCATTTGGAGA-3′, R: 5′-GGGCCTCTTCCTGGTCAATC-3′D2 receptor: F: 5′-GTCTCGTTCTACGTGCCCTT-3′, R: 5′-GGTGGGTACAGTTGCCCTTG-3′D3 receptor: F: 5′-ACTTGGAGGTGACAGGTGGA-3′, R: 5′-GGCATGACCACTGCTGTGTA-3′DAT: F: 5′-CCTGGTTCTACGGTGTCCAG-3′, R: 5′-GCTGACCACGACCACATACA-3′.beta-actin). Each PCR was performed in technical triplicate. Error bars represent the standard error of the mean (SEM) between biological replicates.Relative mRNA levels were quantified using the delta–delta CT method . Outliers were detected using Z scores. Analysis of variance (ANOVA) statistical models were used to examine the effects of between-subject variables such as genotype, sex, and pretreatment. Because of technical limitations, PCR gene expression assay was done separately in samples from male and female mice. Therefore, the statistical analysis could not include a direct male/female comparison but instead focused on genotype and METH pretreatment effects in males and females separately. When significant differences were observed between the genotypes , the analysis of genotype effects was split into D3 receptor subgroups (WT and BDNF HET vs. D3KO and DM) or BDNF genotype subgroups (WT and D3KO vs. BDNF HET and DM) to determine potential genotype interactions and relative contributions to behaviour.Where applicable, a repeated-measures ANOVA was used to analyse within-subject factors in mice’ behavioural data. These included prepulse intensity and drug effects for PPI, time bin analyses and drug effects for locomotor hyperactivity, and change in body weight over the course of the experiment. In cases where data were not normally distributed and sphericity could not be assumed, as demonstrated by a significant Mauchly’s test, the Greenhouse-Geisser correction was used.p < 0.05. Effect size was expressed as partial eta squared (ηp2). Data in all graphs are represented as the mean ± standard error of the mean (SEM).ANOVA results were considered statistically significant when p = 0.006, ηp2 = 0.069), and there was a significant three-way interaction between sex, METH pretreatment, and genotype  = 3.09, p = 0.028, ηp2 = 0.051). To explore this interaction, the data were split by pretreatment and sex and analysed for an effect of the BDNF genotype or D3 receptor genotype , but there was no D3 genotype effect or interaction between the two genotypes. There were no genotype differences in female mice  = 4.30; genotype A,B. Basegenotype A. In conale mice B.p < 0.001, ηp2 = 0.142, not shown). A significant main effect of genotype  = 10.09; p < 0.001, ηp2 = 0.149) prompted further analysis, in which post hoc comparison identified that, independent of METH pretreatment, BDNF HET and DM mice had significantly higher startle compared to D3 knockout mice but not wildtype mice  = 28.62, p < 0.001, ηp2 = 0.400; p = 0.025, ηp2 = 0.052) prompted further investigation of the data split by sex and pretreatment and analysed for an effect of the BDNF genotype or D3 receptor genotype  but there was no interaction with the D3 receptor genotype , independent of genotype, pretreatment, or sex  = 115.4, genotype A,B. In mgenotype A. There genotype A, nor ingenotype B. Compar, or sex C.p < 0.001, ηp2 = 0.579; p = 0.02, ηp2 = 0.055) although the four-way interaction of MK-801 × sex × genotype × pretreatment did not reach statistical significance. However, ANOVA with the genotype split by BDNF heterozygosity or D3 knockout showed that, similar to the APO PPI data, male BDNF HET and DM mice pretreated with METH displayed a significantly attenuated PPI disruption compared to male WT and D3 knockout mice  = 12.02; p = 0.001, ηp2 = 0.218; Compared to acute saline IP injection, acute MK-801 administration significantly reduced PPI  = 237.72, p = 0.009, ηp2 = 0.039; for simplicity shown as sexes combined in p = 0.004, ηp2 = 0.090), an effect which depended on the genotype  = 3.11, p = 0.030, ηp2 = 0.096). Further analysis grouping mice by BDNF genotype showed a significant interaction  = 5.18, p = 0.025, ηp2 = 0.056), and post hoc analysis showed MK-801-induced decreases in startle amplitudes in WT and D3 knockout mice but not in BDNF HET mice and DM mice  = 7.02,  DM mice .p < 0.001, ηp2 = 0.870; p < 0.001, ηp2 = 0.173; p = 0.018, ηp2 = 0.057) suggested sex-specific effects. There were no significant interactions of either the acute or pretreatment effects of METH with genotype in male mice.Compared to the baseline activity displayed after saline injection, a 3 mg/kg METH challenge induced significant locomotor hyperactivity  = 1136.71, p = 0.006, ηp2 = 0.130), prompting further analysis by grouping the animals according to BDNF or D3 receptor genotype. Analysis of the effect of the D3 knockout genotype found no differences between mice with functional D3 receptors (WT and BDNF HET) and D3 knockouts (D3 knockout and DM mice). Conversely, analysis of the BDNF genotype found BDNF haploinsufficiency (BDNF HET and DM) significantly enhanced acute METH-induced locomotor hyperactivity compared to BDNF WT  = 10.46, p = 0.002, ηp2 = 0.105) independent of METH pretreatment  = 4.44, reatment B. This sp < 0.001, ηp2 = 0.371; p < 0.001, ηp2 = 0.141), again suggesting sensitisation to the drug. No significant sex differences were observed, nor genotype differences, in the effect of the 1 mg/kg METH challenge  = 100.26, hallenge C.p = 0.018, ηp2 = 0.252) with further comparisons of the combined genotype groups showing a significantly lower expression in BDNF HET and DM mice compared to wildtype and D3KO  = 10.64, p = 0.003, ηp2 = 0.238). This difference was not seen in female mice , an effect which could be attributed to a lower expression in BDNF HET and DM mice compared to wildtype and D3KO mice  = 12.61, p = 0.001, ηp2 = 0.271). There was also a pretreatment × BDNF genotype × D3 receptor genotype interaction  = 4.67, p = 0.038, ηp2 = 0.121). Further comparison of the data from wildtype and BDNF HET mice showed no differences between the groups. In contrast, irrespective of METH pretreatment, DM mice had slightly lower D2 receptor gene expression than mice with D3 receptor knockout only  = 10.02, p = 0.006, ηp2 = 0.400). There were no differences in D2 receptor expression between the groups in female mice  = 29.9, p < 0.001, ηp2 = 0.461). In addition, D3 receptor gene expression significantly depended on the BDNF genotype  = 8.48, p = 0.006, ηp2 = 0.195; BDNF genotype × D3 receptor genotype, F = 4.62, p = 0.039, ηp2 = 0.117). D3 receptor gene expression was significantly lower in male BDNF HET mice compared to wildtype mice  = 8.76, p = 0.008, ηp2 = 0.316), but there were no differences between D3 knockouts and double-mutant mice, presumably because gene expression was already very low  = 167.5, p < 0.001, ηp2 = 0.827; main effect of BDNF genotype, F = 5.33, p = 0.027, ηp2 = 0.132; D3 genotype × BDNF genotype interaction, F = 4.35, p = 0.044, ηp2 = 0.111). D3 receptor expression was significantly lower in BDNF HET mice compared to wildtype mice  = 6.31, p = 0.021, ηp2 = 0.240) but there were no genotype differences between the D3 knockout groups . Separation of groups by BDNF genotype  = 11.21, p = 0.002, ηp2 = 0.248) showed that the METH pretreatment significantly reduced DAT expression in wildtype and D3KO  = 7.64, p = 0.015, ηp2 = 0.353), but BDNF HET and double-mutant mice tended to show an increase in DAT expression, although this did not reach statistical significance (p = 0.072). In contrast to male mice, in female mice METH pretreatment reduced DAT expression  = 6.16, p = 0.018, ηp2 = 0.153) and this effect differed between the groups  = 3.09, p = 0.040, ηp2 = 0.214). This effect could be attributed to reduced DAT expression depending on the BDNF genotype  = 6.16, p = 0.018, ηp2 = 0.153). A split of the data by BDNF genotype showed that METH pretreatment had no effect in wildtype mice and D3KO, but it significantly reduced expression in BDNF HET and DM mice  = 9.42, p = 0.006, ηp2 = 0.331; While there were no major effects of METH pretreatment on dopamine receptor expression, analysis of DAT expression in male mice G showed p < 0.001, ηp2 = 0.503). Further analysis showed significantly reduced D2 receptor expression in BDNF HET mice and double-mutant mice compared to the combined wildtype and D3 knockout groups  = 27.3, p < 0.001, ηp2 = 0.438) but a significant increase in D3 receptor knockout mice and double-mutant mice compared to wildtype and BDNF HET mice combined  = 10.07, p = 0.003, ηp2 = 0.223). These effects were independent of METH pretreatment . Split analysis of the data by genotype group showed that D2 receptor expression was upregulated by the METH pretreatment in female mice with the D3 receptor knockout genotype  = 19.67, p < 0.001, ηp2 = 0.567) but there was no METH pretreatment effect in wildtype mice and BDNF HET mice  = 37.4, p < 0.001, ηp2 = 0.517). In addition, D3 receptor gene expression significantly depended on the BDNF genotype  = 5.49, p = 0.025, ηp2 = 0.136; BDNF genotype × D3 receptor genotype, F = 4.28, p = 0.046, ηp2 = 0.109). D3 receptor gene expression was significantly lower in male BDNF HET mice compared to wildtype mice F = 6.30, p = 0.021, ηp2 = 0.249), but there were no differences between male D3 knockouts and double-mutant mice, presumably because gene expression was already very low . In female mice, D3 receptor expression was different between the groups  = 14.8, p < 0.001, ηp2 = 0.566) and D3 receptors were markedly downregulated in D3 knockout mice and double-mutant mice  = 44.1, p < 0.001, ηp2 = 0.565), independent of the METH pretreatment  = 16.2, very low E. While reatment F.p = 0.017, ηp2 = 0.257) and specifically the D3 receptor genotype  = 11.7, p = 0.002, ηp2 = 0.257). In male mice with intact D3 receptor expression (wildtype and BDNF HET), METH pretreatment significantly increased DAT expression  = 7.37, p = 0.014, ηp2 = 0.279). In contrast, in male mice lacking D3 receptors (D3 knockout and double-mutant mice), METH pretreatment reduced DAT gene expression  = 4.98, p = 0.041, ηp2 = 0.249). There were no differences between the groups in the frontal cortex of female mice  and mice with the D3 receptor wildtype genotype (BDNF HET and wildtype controls). A previous study described hypersensitivity in D3 knockout mice to amphetamine at a similar dose . HoweverThere were several changes in dopamine receptor and transporter gene expression in the striatum although these generally did not straightforwardly explain the changes in PPI regulation observed in these genotypes. Few previous studies have comprehensively assessed dopamine receptor and DAT mRNA levels following chronic METH, as was done in the present study. We found that METH pretreatment had no long-term effects on dopamine receptor expression in the striatum. In contrast, in male mice, METH pretreatment significantly reduced DAT expression in wildtype and D3 knockout mice, whereas in female mice a decrease in DAT expression was observed only in BDNF HET mice and double mutants. These findings again reveal a strictly sex-specific role of BDNF.Independent of the effect of METH, we observed significantly reduced D1 and D2 receptor expression in male BDNF HET and DM mice compared to wildtype and D3 knockout mice. Male DM mice also had lower D2 receptor gene expression than mice with D3 receptor knockout only. Again, these changes were male-specific, as there were no differences in D1 and D2 receptor expression between the groups in female mice. As expected, D3 receptor expression was significantly reduced in D3KO and DM mice. D3 receptor gene expression was furthermore significantly lower in both male and female BDNF HET mice compared to wildtype mice, consistent with the previously described stimulatory effect of BDNF on D3 receptor expression ,65.Also in the frontal cortex, we found sex-specific downregulation of dopamine receptor gene expression in BDNF HET mice in the frontal cortex. For example, although there were no effects on D1 receptor gene expression in either males or females, in males, a significantly reduced D2 receptor expression was found in BDNF HET mice and DM mice compared to the combined wildtype and D3 knockout groups. In contrast there was a significant increase in female D3 receptor knockout mice and DM mice compared to wildtype and BDNF HET mice combined. While these sex-specific effects were independent of METH pretreatment, in the frontal cortex of female mice D2 receptor expression was upregulated by METH pretreatment in the D3 receptor knockout genotype but there was no METH pretreatment effect in wildtype mice and BDNF HET mice. Finally, as expected, D3 receptor gene expression in the frontal cortex was significantly reduced in D3 knockout mice and in BDNF HET mice compared to wildtype mice. Overall, these data show complex effects of genotype and METH pretreatment strongly dependent on sex.The regulation of PPI involves a complex circuitry with both midbrain and forebrain regions interacting to (1) mediate the startle response, (2) mediate PPI, and (3) modulate PPI mediation . Among tThe mice used in the present study were tested four times for PPI, three times for locomotor (hyper)activity, and subsequently their brains were used for the analysis of dopamine receptor and DAT gene expression. It cannot be excluded that the behavioural tests, including acute drug challenges, influenced the outcome of the PCR analysis. Similarly, it is possible that testing the animals for PPI somehow influenced the outcome of the locomotor hyperactivity tests. Ideally, separate cohorts of mice should have been used for the different behavioural tests, and a separate non-behavioural cohort should have been included for analysis of the effect of METH on dopamine receptor and DAT gene expression. However, this would have required a very much larger number of animals, with the associated ethical and budgetary concerns.We used mice with constitutive gene modifications, and it is possible that these life-long changes in BDNF and D3 expression resulted in compensatory expression changes in other neurotransmitter systems or growth factors. Future studies should therefore include BDNF and D3 receptor gene expression changes at specific later stages of the neurodevelopmental axis. Future studies should furthermore focus on other components of dopaminergic neurotransmission, such as dopamine turnover and metabolism, and differential gene expression effects in neuronal and glial cell populations.These results show an interaction of BDNF with the effects of METH on the regulation of PPI. Specifically, intact BDNF signalling appears to normally protect particularly males from METH causing a dysregulation of PPI. Importantly, our findings do not support a role for D3 receptors in the effect of BDNF. Therefore, the role of these receptors in psychoses and schizophrenia remains unclear. On the other hand, several previous studies have shown reduced levels of BDNF in postmortem brain regions such as the prefrontal cortex and hippocampus ,69,70,71


Recombinant IL-21 protein produced in the CHO-S cells was effective in elevating the expression of antibacterial genes, including β-defensin and lysozyme, and, interestingly, inhibited the NF-κB signaling pathway. Furthermore, we investigated the response of the IL-21 expressing cells to A. hydrophila infection. Immunofluorescent assay showed that IL-21 protein was detected in the CD3γ/δ T cells and was markedly accumulated in the anterior, middle and posterior intestine. Collectively, the results indicate that IL-21 plays an important role in regulating the intestinal inflammation induced by bacterial infection in grass carp.Interleukin (IL) 21 is a pleiotropic cytokine that plays an important role in regulating innate and adaptive immune responses. In fish, the biological functions and cell source of IL-21 remain largely unknown. In this study, we performed qRT-PCR, Western blotting and immunofluorescent microscopy to examine the expression of IL-21 at the mRNA and protein levels. We found that  Interleukin 21 (IL-21) belongs to the gamma chain (γc) family of type I cytokines and possesses a four α-helical structure. It is produced mainly by immune cells, including natural killer T (NKT) cells and activated T cells ,4. IL-21il21 gene was sequenced in the Japanese puffer Fugu rubripesi [il21 genes have been reported in spotted green pufferfish (Tetraodon nigroviridis) [Danio rerio), rainbow trout (Oncorhynchus mykiss) [Paralichthys olivaceus) [Ctenopharyngodon idella) [Channa argus) [il21 differs from that in mammals. Most teleost il21 genes have six exons and five introns whilst mammalian il21 genes have five exons and four introns [il21 gene has the same genomic organization as mammalian homologs. Two IL-21 receptors are present in teleosts and both possess a conserved extracellular cytokine-binding homology domain (CHD), a transmembrane domain and an intracellular domain [In 2005, the first fish ubripesi . Subsequviridis) , zebrafiviridis)  (Danio r mykiss) , Japanesivaceus) , grass c idella) ,20,21 ana argus) . Similara argus) ,17,23. H introns ,17,23. Iil21 has been studied mostly at the transcriptional level. It has been shown that fish il21 genes are constitutively expressed in most tissues but more abundantly in the head kidney, gills and intestine [il21 expression can be modulated by immune stimuli and pathogenic infections. For instance, grass carp and rainbow trout il21 genes have been shown to be upregulated in the primary leukocytes (HKLs) by phytohemagglutinin (PHA) and phorbol myristate acetate (PMA), known to activate T cells, suggesting that il21 could be produced in activated T cells [il21 were identified and could be induced by LPS and polyinosinic–polycytidylic acid (poly(I:C)) in the head kidney leukocytes (HKLs) [il21 expression have been detected in the immune tissues of fish infected with bacterial pathogens such as Yersinia ruckeri (Y. ruckeri), Edwardsiella tarda (E. tarda), Nocardia seriolae (N. seriolae), Aeromonas schubertii (A. schubertii) and Flavobacterium columnare (F. columnare) [il21 is induced by viral hemorrhagic septicemia virus in the kidney of rainbow trout at the early stage of infection [F. columnare using a monoclonal antibody [The expression of fish ntestine ,20,21,22 T cells ,21. In glumnare) ,18,21,22nfection . In our Aeromonas hydrophila (A. hydrophila) induced enteritis is a common disease affecting the grass carp farming industry [A. hydrophila infects a wide range of other fish species, including common carp (Cyprinus carpio), blunt snout bream  and Chinese sea bass (Lateolabrax maculatus), causing intestinal damage and malfunction [Fish are lower vertebrates and have both innate and adaptive immune systems . Like maindustry . In addifunction ,31,32. TA. hydrophila at the transcript and protein levels. Using the monoclonal antibodies previously generated, the IL-21 producing cells were analyzed in different segments of the intestine by immunofluorescent microscopy. In addition, we determined the bioactivity of IL-21 in modulating antibacterial and inflammatory response. Our findings shed light on the functions of IL-21 in regulating the intestinal immunity of bony fish during bacterial infection.In this study, we examined the expression patterns of IL-21 in fish infected with Grass carp (15 ± 2 g) were obtained from Shanghai Binhai Base, Shanghai Ocean University, and acclimatized in a circulating freshwater aquarium at 28 ± 1 °C for at least two weeks before experiments. Fish were anesthetized with MS-222 (100 mg/L) prior to experimental procedures. All experiments were conducted under the national regulations on laboratory animals of China and approved by the laboratory animal ethics committee of Shanghai Ocean University (SHOU-DW-2021-027).Epithelioma papulosum cyprini (EPC) cells  were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum  and 1% penicillin–streptomycin solution (Pen/Strep) at 28 °C in a 5% CO2 incubator [2 shaker. A. hydrophila was provided by the National Pathogen Collection Center for Aquatic Animals, Shanghai Ocean University, China. A. hydrophila was grown at 28 °C in Luria–Bertani (LB) medium in a shaker (180 rpm) until absorbance at optical density (OD) 600 nm reached 0.6.ncubator . CHO-S cil21 (GenBank accession number: KP226585.1) was cloned into pcDNA3.1(+) vector with a 6-histidine tag at the C-terminus of IL-21. The plasmid was termed pcDNA3.1-IL21-His. Il21 promoters  were amplified by PCR and cloned into pGL4.0 vector. All of the primers used in plasmid construction are listed in The open reading frame (ORF) of grass carp ® II 1st Strand cDNA Synthesis SuperMix and Hieff UNICON® Power qPCR SYBR Green Master Mix . ExpiFectamine™ CHO Transfection Kit , jetOPTIMUS Transfection Reagent , lipopolysaccharide . Optimal Cutting Temperature Compound , 4% paraformaldehyde , rabbit polyclonal antibody against β-actin , mouse monoclonal antibody against His-tag , goat α-rabbit or α-mouse IgG secondary antibody , mouse monoclonal antibodies  or CD3γ/δ  were generated and characterized in our laboratory [The main reagents included TRIzolTM Reagent, Hifairboratory . FITC laA. hydrophila were prepared as previously described [7 CFU/mL. Thirty grass carp (15 ± 2 g) were randomly placed into two tanks, each containing 15 fish. Fish were intraperitoneally (i.p.) injected with 100 μL of 1 × 107 CFU/mL A. hydrophila or PBS (control). After 24, 48 and 72 h, peripheral blood leukocytes (PBLs) and immune tissues (25 ± 5 mg), including head kidney, gills, spleen, anterior intestine, middle intestine and posterior intestine were sampled from 4 fish of each group at 24, 48 and 72 h after infection for extraction of total RNA and proteins. In addition, three different intestine segments were sampled at 72 h post-infection and fixed in 4% paraformaldehyde (PFA) for cryosection.escribed , and werA. hydrophila (1 × 107 CFU/mL) or PBS. After 24, 48 and 72 h, anterior, middle and posterior intestine segments were collected and fixed in 4% neutral buffered paraformaldehyde solution for 24 h. The tissue samples were then embedded in paraffin and sectioned at a thickness of 4 μm. Finally, the slides were stained with hematoxylin and eosin (H&E) and photographed under an optical microscope [Grass carp (15 ± 2 g) were i.p. injected with 100 μL croscope .A. hydrophila were prepared as previously described [600 of A. hydrophila culture reached 0.6, 1 mL of bacteria were collected and washed three times with sterile PBS. Bacterial cells were resuspended with 1 mL sterile PBS and placed in a water bath at 100 °C for 30 min. The inactivated A. hydrophila were stored at 4 °C. Grass carp primary HKLs were prepared as previously described [7 cells/mL). After 6 h cultivation, HKLs were stimulated with inactivated 100 μL A. hydrophila (1 × 107 CFU/mL) or LPS (75 μg/mL). Control cells were treated with PBS. Cells were collected at 12 h and 24 h for qRT-PCR and Western blotting analysis. In addition, HKLs were stimulated with inactivated A. hydrophila, LPS or PBS for 12 h and 24 h, and fixed with 4% PFA for immunofluorescent microscopy.Heat-inactivated escribed . In brieescribed  and seedTM reagent. The first-strand cDNA was synthesized using the Hifair® II 1st Strand cDNA Synthesis SuperMix and stored at −80 °C. qRT-PCR was performed using Hieff UNICON® Power qPCR SYBR Green Master Mix and the LightCycler 480 Instrument . qRT-PCR was performed using the following conditions: 1 cycle of 95 °C for 30 s, 40 cycles of 95 °C for 5 s, 62 °C for 30 s, 72 °C for 10 s, followed by 1 cycle of 95 °C for 10 s, 65 °C for 60 s, 97 °C for 1 s. qRT-PCR was set up as follows: 5 μL SYBR® Green PreMix Ex Taq™ II , 1 μL cDNA template, 0.2 μL forward primer (10 μM), 0.2 μL reverse primer (10 μM), and 3.6 μL H2O. The primers used for qRT-PCR assay in this study are listed in ef1α) gene was used as reference gene for normalization of gene expression. Data were analyzed using the 2−△△Ct method [Total RNA was extracted from tissues (25 ± 5 mg) and cell samples using TRIzolt method .TM reagent. In brief, the protein pellet (white) was collected between the aqueous and organic phases, briefly dried and resuspended in 300 μL absolute ethanol. The protein solution was placed at room temperature for 2–3 min before centrifugation at 2000× g at 4 °C for 5 min. The upper organic phase was carefully transferred to a fresh centrifuge tube, and gently mixed with 1.5 mL isopropyl alcohol, followed by incubation at room temperature for 10 min. Protein pellet was obtained by centrifugation at 12,000× g at 4 °C for 10 min, washed three times with 0.3 M guanidine hydrochloride, resuspended in 2 mL ethanol for 20 min, and dissolved in 1% SDS (sodium dodecyl sulfate) at 50 °C. The protein solution was centrifuged at 12,000× g at 4 °C for 10 min and stored at −20 °C before use.Proteins used for Western blotting were extracted from the intermediate protein layer between the aqueous and organic phase in RNA extraction using TRIzolv/v, 5 μg/mL) at 4 °C overnight. After washing with TBST three times, the membrane was incubated with secondary antibodies  for 1 h and washed extensively with TBST. The membrane was photographed using the Odyssey CLx Imaging System .Western blotting analysis was performed. Briefly, the protein samples were dissolved in lysis buffer at 4 °C for 30 min. Subsequently, protein solution was mixed with 5×SDS protein loading buffer and boiled for 10 min. Protein solution was separated on a 12% SDS-PAGE gel and transferred onto a polyvinylidene difluoride (PVDF) membrane  using a semidry transfer unit . The membrane was blocked with TBST buffer containing 5% skimmed milk for 1 h, followed by incubation with primary antibodies  in darkness at 4 °C overnight and incubated with 1 μg/mL 4,6-diamidino-2-phenylindole  in darkness for 10 min. The coverslips were observed under a Leica confocal microscope  and photographed.Freshly prepared primary HKLs were washed with PBS, cytospinned onto a coverslip, and fixed with 4% PFA for 20 min. Cells were then permeabilized with PBS containing 0.3% Triton X-100 for 15 min, and blocked in PBS containing 5% bovine serum albumin (BSA) for 1 h. Coverslips were overlaid with the FITC-labeled GC9-IL21 mAb  and GC38T-Cy5 mAb  at 4 °C overnight. After washing, slides were stained with DAPI and photographed under a Leica confocal microscope.Immunofluorescent microscopic analysis of tissues was carried out as previously described . In brie2 until cell numbers reached 6 × 106 cells/mL. Twenty-five µg pcDNA3.1-IL21-His plasmids were diluted with 1 mL OptiPRO medium containing 80 µL ExpiFectamine™ CHO Reagent and 920 µL OptiPRO medium. After a 5 min incubation, the mixture was added into the flask containing CHO-S cells (6 × 106 cells/mL), and after 18 h, 150 µL ExpiFectamine CHO Enhancer and 6 mL ExpiFectamine CHO Feed were added. Five days later, the same volume of ExpiFectamine CHO Enhancer and ExpiFectamine CHO Feed were added again. At day 8 post-transfection, culture media containing recombinant proteins were collected and applied for protein purification. The purity and size of proteins were verified by SDS-PAGE and Western blotting, which are described below. Purified proteins were aliquoted and stored at −80 °C until use.Recombinant IL-21 (rIL-21) protein was expressed in CHO-S cells and purified. In brief, CHO-S cells were cultured in a 37 °C shaker (125 rpm) supplemented with 8% CO2 incubator. The rIL-21 protein was added into the cells at final concentrations of 5, 50, and 100 ng/mL. Control cells were treated with PBS. After 24 h, the cells were collected for qRT-PCR analysis.To determine the bioactivity of the rIL-21 protein, primary HKLs of the grass carp were prepared according to our previous study . The celil21 promoter plasmids or pGL4.0 (500 ng) were co-transfected with pRL-TK (50 ng) using jetOPTIMUS Transfection Reagent. The transfected cells were treated with inactivated A. hydrophila, LPS or PBS and harvested at 12 h and 24 h for promoter activity analysis. Alternatively, EPC cells were co-transfected with NF-κB reporter plasmid (500 ng) and pRL-TK (50 ng) using jetOPTIMUS Transfection Reagent. After 24 h, the cells were incubated with PBS or different concentrations of rIL-21 protein  for 24 h. The cells were collected and lysed with passive lysis buffer at 4 °C for 30 min, and the luciferase activity was measured following the manufacturer’s protocol.EPC cells have been previously shown to have high efficiencies for plasmid transfection and commonly used for in vitro studies . Therefot-test. p < 0.05 and p < 0.01 were considered significantly different.Data were statistically analyzed using the SPSS 22.0 software. The statistical significance of biotesting of rIL-21 protein was analyzed for one-way ANOVA and mean comparisons between tested groups were conducted using Duncan’s new multiple range test (DMRT). The statistical significance of other data between the treatment and control groups was determined by Student’s A. hydrophila. As shown in A. hydrophila infection resulted in damage to the microvilli, massive infiltration of leukocytes and increases of goblet cells in all three intestinal segments  were used as controls to exclude false positive cells  and 24 h (p < 0.05 or p < 0.01) (A. hydrophila were more effective than LPS in inducing the expression of il21 (+ cells markedly increased in the HKLs after stimulation (Head kidney (HK) is the hemopoietic organ producing immune cells in fish. To examine IL-21 response to stimulation with LPS and inactivated  < 0.01) A,B. It a of il21 B. Consismulation C,D. Isotmulation .il21 expression, we constructed 3 plasmids containing the promoter regions of il21 gene . Similarly, il21 pro-1 and il21 pro-2 were activated at 24 h post stimulation (p < 0.05 or p < 0.01). Furthermore, dose-dependent effects of activation by LPS and inactivated A. hydrophila were apparent (p < 0.05 or p < 0.01)  D,E. Convimulants B,C. Takeβ-defensin 1, β-defensin 2 and β-defensin 3 were induced by rIL-21 at the doses of 50 and 100 ng/mL (p < 0.01) and that c-lysozyme was upregulated by 100 ng/mL rIL-21 (p < 0.05) , il21r (p < 0.05) and il10 (p < 0.01) (tnfα (p < 0.05)  (p < 0.05 or p < 0.01)  D. Howevenaltered E. In add < 0.01) F–H but d < 0.05) J. The ex5) (il1β I was not < 0.01) K,L. We a < 0.01) M. These il21 genes are expressed mainly in the head kidney, spleen and mucosal tissues including gills and intestines, and are inducible in response to immune stimuli and bacterial infections [il21 expression was detected in the spleen and head kidney of fish infected with Y. ruckeri and viral hemorrhagic septicemia virus [E. tarda [A. schubertii or N. seriolae [A. hydrophila . These results suggest that IL-21 exerted anti-inflammatory functions by inhibiting the NF-κB pathway in fish. In support of this notion, it has been shown that mouse IL-21 suppresses NF-κB signaling induced by LPS in peritoneal macrophages [We determined the biological activities of grass carp IL-21 . Akin toammation ,19,21,43ary HKLs ,19. Intrt manner  but unexg-lysozyme) and chicken-type lysozyme (c-lysozyme), have been identified [c-lysozyme but not g-lysozyme was increased in the HKLs after stimulation with IL-21  where T lymphocytes and other immune cells reside for interaction with each other . Grass ctalities . On the ubmucosa . Interesammation ,48,49 anammation ,51. It i+ T cells and NKT cells [il21 were detected in the HKLs and splenocytes following stimulation with PHA and PMA [+ T cells. The IL-21 producing CD3γ/δ+ T cells were found mainly in the submucosa and were induced in the anterior, middle and posterior intestine after A. hydrophila infection (+ T cells could play important roles in the maintenance of immune homeostasis in fish and the control of excessive inflammation during infections.In mammals, IL-21 is produced by activated CD4KT cells . In rain and PMA ,21, bothnfection . It is eil21 was induced during infection with A. hydrophila. Recombinant IL-21 protein was effective in upregulating antibacterial genes, including β-defensins and lysozyme. In addition, the IL-21-expressing CD3γ/δ T cells were detected in tissues and markedly increased in the anterior, middle and posterior intestine after infection with A. hydrophila. Our results suggest that IL-21 plays an important role in regulating intestinal inflammation induced by bacterial infection (In summary, we have shown that grass carp nfection .A. hydrophila infection. Moreover, the recombinant IL-21 protein was effective in elevating the expression of antibacterial genes and inhibiting the NF-κB signaling pathway. The results demonstrate that IL-21 is involved in regulating the intestinal inflammation induced by bacterial infection in grass carp.In conclusion, this study provides evidence that IL-21 is induced and markedly accumulated in the CD3γ/δ T cells of the anterior, middle and posterior intestine of grass carp during


Five coordination polymers were obtained based on an azo multifunctional aromatic ligand. The movement and collision behaviors of components are greatly affected by the magnetic field assisted method, which could have a subsequent impact on their structures with different space groups. 1(absa)(H2O)4]n and [TM2(absa)(bipy)(H2O)]n  were synthesized by solvent evaporation under a magnetic field. It is evident that magnetic fields bring significant and noticeable changes to the absa2− ligand orientation and the component movement behaviors to construct coordination polymers. The absa2− ligands bind to the metal ions in bridging coordination mode through the carboxylate groups, in addition to the bipy molecules adopting bridging modes. Photoluminescence measurements indicate that the emissions of compounds 1–5 are at 626, 600, 632, 658 and 682 nm in the solid state, respectively.Five coordination polymers [TM When the Na2absa ligand was used in combination with the rigid 4,4′-bipyridine (bipy) building block, five transition metal coordination polymers were synthesized via solvent evaporation under a magnetic field (see below).Herein, Na2.2.1.Kα radiation (λ = 0.71073 Å) using either the ω or the φ–ω scan mode. Elemental analyses of carbon, hydrogen and nitrogen were performed with a Perkin Elmer 240C elemental analyzer. The infrared spectra were measured by a Magna-IR 750 spectrophotometer in the 4000–400 cm−1 region (KBr pellet). Thermogravimetric analyses (TGA) were carried out on a NETZSCH STA 449C unit at a heating rate of 10°C min−1 under a nitrogen atmosphere. Photoluminescence analyses were performed on a Perkin Elemer LS55 fluorescence spectrometer.The reagents and solvents employed were commercially available and used as received without further purification. Single-crystal X-ray diffraction data were collected with a Rigaku Saturn 70 CCD, a Bruker APEX-II diffractometer or a Bruker D8 VENTURE TXS PHOTON 100 equipped with graphite monochromated Mo 2.2.2absa) was synthesized using procedures described in the literature  with stirring to a pH value of 5, and the reaction mixture was further stirred for 1 h at room temperature. The product was recrystallized three times and colorless crystals were collected by filtration and dried under vacuum at room temperature.5,5′-azobissalicylic acid (H2.3.2absa (0.1 mmol) were added to a vessel containing mixed solvent (5 ml of methanol and 10 ml of distilled water). The mixed solution was stirred and heated at 100°C for 3 h under a 1 T magnetic field. The solution obtained was kept in a small vial covered with parafilm at room temperature and left under a magnetic field. Block crystals were obtained after 10 days. For compound 1, IR : 3023, 1579, 1479, 1448, 1328, 1248, 1187, 1079, 828, 789, 720, 577, 473. Calculated for C14H16N2O10Zn (%): C, 38.39; H, 3.66; N, 6.40. Found (%): C, 38.42; H, 3.82; N, 6.46. For compound 2, IR : 3211, 1577, 1330, 1481, 1450, 1385, 1249, 1187, 1140, 1079, 853, 826, 792, 718, 677, 626, 575, 473. Calculated for C14H16N2O10Co (%): C, 38.96; H, 3.71; N, 6.49. Found (%): C, 38.90; H, 3.80; N, 6.55.The corresponding metal nitrates (0.1 mmol) and Na2.4.3–5 were synthesized via a similar procedure used to produce compound 1 with the corresponding metal nitrates (0.1 mmol), Na2absa (0.1 mmol) and bipy (0.1 mmol). For compound 3, IR : 3064, 1611, 1569, 1483, 1426, 1385, 1362, 1293, 1258, 1220, 1187, 1073, 832, 805, 783, 722, 642, 587, 457. Calculated for C24H18N4O7Zn (%): C, 53.35; H, 3.33; N, 10.37. Found (%): C, 53.41; H, 3.40; N, 10.35. For compound 4, IR : 3276, 1591, 1560, 1444, 1352, 1265, 1211, 1063, 807, 673, 630, 587, 465. Calculated for C24H18N4O7Co (%): C, 52.23; H, 3.63; N, 10.16. Found (%): C, 52.20; H, 3.65; N, 10.25. For compound 5, IR: 3066, 1569, 1477, 1436, 1379, 1328, 1254, 1185, 1071, 807, 681, 638, 587, 477. Calculated for C24H18N4O7Cu (%): C, 53.54; H, 3.35; N, 10.41. Found (%): C, 53.57; H, 3.46; N, 10.65.Compounds 2.5.1, 6 and 7, the measurement device used was a Rigaku Saturn 70 CCD; scan mode: ω; data collection, cell refinement and data reduction were carried out using CrysAlisPro . The data can be obtained free of charge from 3.3.1.1 and 2 have similar structures, although their space groups and unit cells are different. Therefore, only the structure of compound 1 is described in detail. Compound 1 crystallizes in the space group P21/c with the monoclinic system (Table S1 of the supporting information). The asymmetric unit comprises one Zn atom, one absa2− ligand and four coordinated water molecules [Fig. 1a)]. Each Zn atom is in a distorted octahedron [Fig. 1b)]. The absa2− ligands bridge with the Zn ions to generate a chain structure through the carboxylate groups in a monodentate coordination fashion [Fig. 1c)]. Note however that both phenol groups of the absa2− ligand are not coordinated to Zn ions which, in the protonated state, balance the charges in compound 1.Single-crystal X-ray analysis reveals that compounds 3–5 are similar, hence only compound 3, as an example, is described in detail. Single-crystal X-ray analysis revealed that compound 3 crystallizes in the triclinic space group P1 (Table S3). Its asymmetric unit consists of one crystallographically independent Zn atom, one absa2− ligand, one bipy molecule and one coordinated water molecule [Fig. 2a)]. Each Zn atom adopts a distorted square-pyramidal coordination geometry [Fig. 2b)]. The bipy molecule binds to the Zn ions, acting as a typical bridging ligand (Table S4). Each absa2− ligand binds to Zn ions in bridging mode through both monodentate carboxylate groups, leaving both protonated phenol groups uncoordinated. Each bipy molecule bridges with Zn ions to form a wave-like one dimensional chain structure [Fig. 2c)], and the absa2− ligands link these one-dimensional chains to generate a two dimensional network through the coordination of carboxylate groups with Zn ions [Figs. 2d) and 2e)].The structures of compounds 2− and two bipy ligands, and thus can be simplified as four-connected nodes, with the absa2− and bipy ligands acting as connecting rods. The overall topology can be described as a four-connected framework [Fig. 2f)]. From a topological point of view, it exhibits a two-dimensional layered net with the Schläfli symbol (43·63).To gain better insight into the framework structure, a topological analysis was carried out. Zn atoms bind to two absa3.2.1–5 were synthesized by the application of a magnetic field. Compounds 1 and 2 have similar one-dimensional structures, which crystallize in the space group P21/c with the monoclinic system, and space group  P43212 with the tetragonal system, respectively. Compounds 3–5 have similar two-dimensional frameworks, which crystallize in the triclinic space group P1, and monoclinic space group P21/n. Note that their architectures are similar, but there is a slight difference in the lattice packing. Magnetic fields can cause the interesting phenomena observed in this work. Therefore, it is necessary to compare the magnetic field effects in the packings and framework geometries. As reported in the literature, on the one hand, the presence of a magnetic field has a significant influence on the intermolecular interactions of coordination polymers , and 2.012 Å (Zn—O) and 2.114 Å (Co—O) in compounds 3 and 4 (Table S4), respectively. Magnetic fields can strengthen the bonding interaction through induction interactions ·(bipy) (6) is separated from the solution, where the absa2− ligand and bipy molecule are uncoordinated from the metal ions [Fig. 3a) and Table S5]. This may be attributed to the fact that the carboxylate groups in the H2absa ligand are protonated and bind to fewer metal centers.In order to better explain the result, we conducted comparative experiments. Under zero magnetic field, 3]·(absa)·7H2O (7) was obtained [Fig. 3b) and Table S5]. Under a 1 T magnetic field, the absa2− ligands can resist irregular movement to a certain extent, and tend to be perpendicular to the magnetic field. Thus, they gain the opportunity to approach metal ions to form one-dimensional chains. When bipy is added under magnetic field, the absa2− ligand is perpendicular to the magnetic field, which makes the ligand coordinate with metal ions in a certain direction, generating a two-dimensional net.With the increase of the magnetic field to 0.3 T, phen molecules, acting as chelated terminated ligands, prefer to coordinate with metal ions and occupy all coordination sites, in contrast to absa1–5 were isolated. Many attempts were made to obtain more crystals by improving the reaction conditions, but were unsuccessful. Because not enough samples were available, additional measurements were not performed, but thermogravimetric analyses and photoluminescence properties were studied. The effect of magnetic field on the types of structures are rather complicated and difficult to discuss. However, it is worth noting that the metal sources and the auxiliary ligands also play an important role. The orientation of the absa2− ligand in a magnetic field will affect its coordination mode in compounds 1 and 2 relative to compounds 6 and 7. The presence of a high magnetic field and bipy facilitates the formation of high-dimensional coordination polymers such as compounds 3–5. However, much more systematic work is needed to further elucidate the magnetic field effect, which can influence and induce the formation of the resultant coordination compounds. It is also anticipated that further new types of coordination polymers can be designed by this synthetic method.Very few block crystals of compounds 3.4.1–5, TGA was performed under a nitrogen atmosphere at the heating rate of 10°C min−1 between 25 and 900°C . The second weight losses in the temperature ranges 156–900°C and 189–900°C correspond to the pyrolysis of the absa2− ligands. Up to 900°C, the thermogravimetric curves still show downward trends. The residues may be the metal oxide. For compounds 3–5, the weight losses in the temperature ranges 25–315°C, 25–220°C and 25–237°C amount to 3.4, 6.7 and 3.4%, which can be attributed to the removal of the coordinated water molecules . The weight losses of 29.0, 28.5 and 29.5% in the temperature ranges 315–371°C, 220–313°C and 237–280°C correspond to the loss of the bipy molecules . The weight losses in the temperature ranges 371–900°C, 313–900°C and 280–900°C correspond to the decomposing of the absa2− ligands. Up to 900°C, the thermogravimetric curves still show downward trends. The residues may be the metal oxide.To investigate the thermal stability of compounds °C Fig. 4. The TGA3.5.et al., 20091–5 as well as the free Na2absa ligand for comparison were measured at room temperature  compound_1, compound_2, compound_3, compound_4, compound_5, compound_6, compound_7. DOI: 10.1107/S2052252523007650/ed5029sup2.pdfSupporting tables. DOI: 2210140, 2210141, 2210142, 2210143, 2210144, 2210145, 2210146CCDC references: