Penicillium expansum for which such a target has not yet been achieved. Accordingly, no point-of-need tests commonly used in food immunodiagnostics are commercially available for patulin. In the present study, three functionalized derivatives conforming to generally accepted rules in hapten design were firstly tested to generate suitable antibodies for the sensitive immunodetection of patulin. However, these conventional bioconjugates were unable to elicit the desired immune response, so an alternative strategy that takes advantage of the high electrophilic reactivity of patulin was explored. Patulin was reacted with 4-bromothiophenol, and the obtained adduct was used to produce antibodies with nanomolar affinity values. These results demonstrated for the first time that targeting the adduct resulting from the reaction of patulin with a thiol-containing compound is a promising approach for developing user-friendly immunoanalytical techniques for this elusive mycotoxin.Mycotoxins represent a major concern for human and animal health because of their harmful effects and high occurrence in food and feed. Rapid immunoanalytical methods greatly contribute to strengthening the safety of our food supply by efficiently monitoring chemical contaminants, so high-affinity and specific antibodies have been generated for almost all internationally regulated mycotoxins. The only exception is patulin, a mycotoxin mainly produced by  According to the RASFF annual reports of the European Commission, around 45% of the informative notifications issued in recent years were related to the presence of chemical substances such as pesticides, biotoxins, veterinary drugs, food additives, etc.2. Among them, mycotoxins constitute the group of chemical hazards that is responsible for the highest number of notifications, which translates into economic losses for the agri-food industry worth millions of Euros per year. Therefore, strict regulatory guidelines have been established internationally for the major classes of mycotoxins3.The presence of toxic chemicals in food, particularly in products intended for children's consumption, is a matter of great concern to consumers and international health authoritiesPenicillium expansum4. Different studies in rodents have established that long-term exposure to patulin leads to neurotoxic, immunotoxic, genotoxic, and teratogenic effects. However, the International Agency for Research on Cancer (IARC) has classified patulin in Group 3 as not carcinogenic to humans because of insufficient evidence6. The main route of human exposure to patulin is through the ingestion of contaminated fruits, mainly apples and their derived products, like juices and compotes. Patulin is highly stable during food processing, and available technologies aimed at removing or reducing patulin from contaminated juices has been of limited efectiveness so far8. The European Union has established maximum allowable levels of patulin in juices (50 µg/kg), apples (25 µg/kg), and in foodstuffs intended for consumption by infants and children (10 µg/kg), and similar criteria were adopted by other countries. International studies on the incidence of patulin in food commodities have shown that a high percentage of the analysed samples contained this mycotoxin11. More importantly, a series of alerts and product recalls have emerged due to the presence of patulin at levels exceeding the regulatory limits in apple-derived products in the European Union, the United States, Canada, Australia, and Hong Kong, among others12.Patulin [4-hydroxy-4H-furo[3.2-c]pyran-2(6H)-one] Fig.  is a toxi.e., those based on the use of antibodies as specific recognition elements, are widely implemented in laboratories worldwide, and a high number of immunodiagnostic companies are commercializing rapid tests for the control and detection of regulated mycotoxins, with the remarkable exception of patulin. As a result of the lack of point-of-need methods, the analysis of patulin is mainly carried out by instrumental chromatographic techniques14.In the field of mycotoxin monitoring, immunoanalytical techniques, 15. That strategy consisted of introducing a carboxyl linker at the C-4 position of the molecule by reacting patulin with succinic anhydride; however, low antibody titres were obtained and poor recognition of free patulin was observed. This approach was further followed by other authors, with similar disappointing results18. In 2007, de Champdoré et al. reported a different strategy consisting of attaching the spacer arm at the C-7 atom of the patulin framework19, but the ability of the so-obtained antibodies to recognize free patulin in solution by standard competitive ELISA was not demonstrated. Accordingly, it remains controversial whether the approaches described to date for the generation of anti-patulin antibodies may result in specific, high-affinity binders suitable for the development of rapid test formats commonly used in food immunodiagnostics, like ELISA and immunochromatographic strips. Moreover, the lack of commercial immunoassays for patulin most likely reflects the inherent difficulties in raising antibodies for this relevant mycotoxin.The first reported attempt to produce anti-patulin antibodies dates back to 1986C4 and C7 in this work were those previously proposed by the above-mentioned authors, while hapten C6 is first reported herein. These three haptens constituted what may be called the conventional approach, because the reasoning behind their synthesis was to introduce the linker at selected sites of the molecular framework in order to cause minimum disturbances in the conformation and the electronic distribution of the molecule. Finally, a disruptive novel strategy harnessing the high electrophilic reactivity of patulin was additionally explored in order to trigger the immune response to this elusive mycotoxin.The aim of the present study was to generate high-affinity antibodies enabling the sensitive immunodetection of patulin. To this purpose, three functionalized derivatives of the mycotoxin were synthesized and covalently coupled to carrier proteins. All three haptens Fig.  were rat21. Accordingly, previous attempts have tried to adhere strictly to this general rule. The approach to this problem that was originally proposed by Mehl et al. consisted of the incorporation of the spacer arm through the hydroxyl group that is naturally present in patulin via O-acylation. This strategy theoretically grants a proper exposure of the mycotoxin to the immune system while causing minimum modifications in the analyte structure15. On the other hand, the strategy suggested by de Champdoré et al. consisted of introducing the carboxylic linker at position C-719. This derivative displays clear structural differences with respect to patulin. The introduction of the linker at this position is at the expense of suppressing the C7-C7a double bond, and this modification leads to a substantial deviation from the quasi-coplanarity of the patulin framework due to the pyramidalization of both positions—the sp2-trigonal hybridization changes to sp3-tetrahedral. The authors justified their choice in an attempt to enhance the stability of the derivative by decreasing the reactivity of the molecule to nucleophiles.Patulin monitoring by antibody-based rapid methods is a long-standing goal in food immunodiagnostics, but the small size of the molecule together with its high electrophilic reactivity makes the aim of obtaining suitable antibodies certainly a challenge. Common knowledge in the field of immunoanalytical methods to haptens advices that the functionalized immunizing derivative should closely mimic the conformation, main chemical groups, and electronic distribution of the target moleculeC4 and C7, which were synthesized as summarized in Fig. S)-patulin methyl ether (6) was obtained with an overall yield of 44.6% starting from L-(+)-arabinose, essentially as previously reported22. From this derivative, hapten C4 was obtained in two straightforward steps with a yield of ca. 54%, whereas hapten C7 was attained with a yield of ca. 22%, also in two steps.The two above-mentioned approaches are herein represented by haptens C6 in Fig. C6 share the same skeleton and functional groups. Additionally, the introduction of the hydrocarbon chain through the position C-6 implies a notable increase in steric hindrance near the adjacent unsaturated position (C-7), thus resulting in a reduced susceptibility of C-7 to nucleophilic addition, which is deemed the main cause of the remarkable reactivity of patulin.With the aim of attaining the lowest possible structural modification of patulin, an alternative approach was envisioned (hapten 9) , the primary hydroxyl group was selectively desilylated (11) and converted to the corresponding aldehyde (12) by oxidation with tetrapropylammonium perruthenate (TPAP) and N-methylmorpholine-N-oxide (NMO). The completion of the carboxylated spacer arm (as methyl ester) first involved Wittig reaction with the stabilized ylide (carbomethoxymethylene)triphenylphosphorane, followed by hydrogenation of the generated double bond under homogeneous conditions using Wilkinson’s catalyst. Once the insertion of the linker was accomplished (14), the preparation of the common hapten core was carried out starting with the deprotection of the hydroxyl groups under mild conditions (15). The hydroxyl groups at the C-4 and C-5 positions were then selectively re-protected by the formation of an isopropylidene ketal (16), thus allowing the further oxidation of the remaining hydroxyl group with TPAP-NMO. Wittig olefination of the obtained ketone (17), using the same ylide as above, afforded a 5:1 mixture of E/Z stereoisomers (18) that could not be separated by column chromatography, but that after the hydrolysis of the isopropylidene group under acid conditions led directly to the unsaturated γ-lactone 19 in 69% global yield from 17. Transformation of the axially disposed hydroxyl group at the C-7 position into a mesylate leaving group allowed for the formation of the C7‒C7a double bond via a bimolecular β-elimination reaction (E2), thus completing the functionalization of the hapten framework characteristic of patulin (20). Finally, hydrolysis of the methyl ester group under mild conditions, using a lipase from Candida antarctica immobilized on an acrylic resin, followed by acid-catalysed hydrolysis of the acetal methoxy group of 21, afforded the desired hapten C6 as a 2:1 mixture of epimers at the hemiacetal carbon. The overall yield of the 13-steps sequence leading to the hapten C6 was about 11%.The synthetic route for the preparation of this hapten started from commercially available methyl α-D-galactopyranoside (9) Fig. . After pN-hydroxysuccinimide (NHS). The thus obtained N-hydroxysuccinimidyl esters of haptens C4 and C6, C4–NHS and C6–NHS, respectively, were isolated and purified prior to their conjugation to the carrier proteins in order to prevent undesired secondary reactions and to achieve a fine control over coupling conditions. The structure of both active esters  was confirmed by 1H NMR spectroscopy. In the case of hapten C7 we observed a significant loss of the linker during the activation step that led to a mixture of the corresponding active ester (C7-NHS) and patulin—up to 30%, which increased notably when trying to separate it from the active ester by column chromatography. The easy loss of the spacer arm in hapten C7—a bimolecular E2 elimination process—can be attributed to the anti-coplanar relationship that exists between the (3-carboxypropanoyl)oxy moiety at C-7 (a relatively good leaving group) and the hydrogen atom at the C-7a allylic position. Given the difficulties encountered for the isolation and chromatographic purification of C7–NHS, it was used without further purification for the preparation of bioconjugates. It must be noted that the instability of the linker is a matter of concern when dealing with hapten activation and conjugation because it may occur later in vivo following inoculation with the bioconjugate.As shown in the previous figures, all three haptens were equipped with a free carboxylic acid at the end of the linker, so they could be readily activated through carbodiimide-mediated esterification with C4, C6, and C7, respectively, whereas the OVA bioconjugates had values ranging from 2 to 7. It is worth noting that the coupling yields for hapten C7 were substantially lower, which is likely due to the aforementioned ease with which the elimination of the spacer arm occurs in this hapten.Bovine serum albumin (BSA) and ovalbumin (OVA) were chosen as carrier proteins. The activated haptens were added to BSA and OVA at 30-fold and 10-fold molar excess, respectively. The bioconjugates were dialysed after size-exclusion chromatography purification, and the final hapten densities were measured by MALDI-TOF analysis. In the case of BSA Fig. A, the fo. addressed the production of antibodies to patulin, they introduced the linker at the C-7 position of the molecule because of the high electrophilic character of the mycotoxin. The authors wisely reasoned that the reactivity of the molecule would be seriously compromised if the double bond in the dihydropyran ring was removed. However, attending to the outstanding previous works by Fliege and Metlzer, who first reported a comprehensive study on the strong reactivity of patulin towards nucleophiles24, this scenario seems to be only partially true. Thus, when patulin reacts with a thiolated compound, an addition to the C-7 position does happen first, but the reaction does not stop at this point. On the contrary, the resulting derivative is still able to react with additional nucleophiles giving rise to a high number of different compounds. Therefore, the studies by Fliege and Metlzer provide a feasible explanation of why haptens C4, C6, and C7 were not able to elicit the production of anti-patulin antibodies with desirable binding features. Even if the NHS esters of the haptens are purified and characterized before protein coupling, and conjugates are unequivocally identified by MALDI-TOF spectrometry, as it was done in this study, haptens grafted in the conjugate may react in vivo with biological S-nucleophiles (and even N-nucleophiles) following inoculation of experimental animals26. These reactions would lead to a severe modification of the molecular structure of the hapten, and therefore antibodies unable to recognize the intact mycotoxin would be generated.The polyclonal antibodies derived from rabbits immunized with BSA–hapten conjugates were assayed by indirect competitive ELISA. Low-to-moderate titres were observed, and none of the six antibodies showed significant recognition towards free patulin in solution, even at a concentration of 10 µM. When de Champdoré et al29. Encouraged by those studies, this antibody—kindly supplied by the company − was tested using our collection of bioconjugates. Antigens OVA–C4 and OVA–C7 were recognized by this antiserum, although with low titres. However, no inhibition by patulin in solution was either detected in competitive assays. To gain further insights into the binding properties of this antibody, OVA was modified with succinic anhydride, the spacer arm used in haptens C4 and C7. This pseudo-conjugate, with no patulin attached, was also able to interact with the antibody, indicating that it may actually be recognizing the spacer arm that links the hapten to the carrier. These results suggest that haptens C4 and C7 could have partially lost the patulin skeleton during conjugation and/or immunization, leaving exclusively the succinyl chain bound to the protein. This possibility is supported by our observations during the synthesis and activation of hapten C7 and previous works on the reaction of acetyl patulin  with nucleophiles23.Despite no commercial rapid tests for patulin immunodetection are available, several authors have reported the use of a rabbit polyclonal antibody from Agrisera to develop rather complex immunoanalytical strategies for the determination of patulin31. Such conclusion not only applies to haptens C4 and C7 with a succinyl spacer arm, but also to hapten C6, a derivative that was synthesized through an elaborated synthetic pathway and that does not raise doubts about its stability during purification and conjugation.Overall, these experiments strongly support the idea that the generation of anti-patulin antibodies through conventional approaches aimed at preserving the structural integrity of the mycotoxin is not probably feasible. The high reactivity of the mycotoxin to nucleophiles, combined with the small size of the molecule and the instability of the hapten, either in vitro or in vivo, are factors likely underpinning this conclusion. In fact, the toxicity of the mycotoxin relies on its capacity to modify cysteine residues in proteins/enzymes, thus inactivating or reducing their activity and affecting the function of essential cellular systems, directly or indirectly23. The determination of a derivative of the analyte instead of the analyte itself has already been used successfully for other very small chemicals such as glyphosate and acrylamide33. For such an approach to be of practical interest, certain requirements must be met. Thus, (i) the reaction of patulin and the thiol should take place in an aqueous medium; (ii) the reaction should proceed through fast reaction kinetics so the product is formed within minutes rather than hours or days; (iii) the reaction should proceed reproducibly and in high yields, preferably quantitatively; and (iv) the reaction should result in a major compound, ideally a single one. While all of these requirements are important, the last one is of paramount relevance. Previous studies clearly showed that the reaction of patulin with thiols yields a large number of products through complex reaction pathways starting with a Michael-type addition to the electrophilic C-7 position of the α,β,γ,δ-unsaturated lactone. In particular, the reaction of patulin with 4-bromothiophenol allowed to identify up to sixteen different adducts23, far from the desirable goal of obtaining a single compound. However, careful examination of these results showed that five of the sixteen resulting adducts were formed because methanol was used as a co-solvent, while other products were formed under conditions where the thiol was present in high excess and/or after long reaction times. More importantly, these experiments also showed that one predominant compound was formed, namely the adduct formed by the incorporation of two thiol molecules into the mycotoxin scaffold at positions C-7 and C-4. Another key condition for the success of the derivatization strategy is that the final structure should be complex enough to elicit an appropriate immune response. Therefore, aromatic thiol-containing compounds seem to be more suitable as derivatization reagents than aliphatic thiols. Taking all these considerations into account, different reaction conditions were investigated to improve the formation of a di-thiol adduct in the absence of methanol and using six simple and commercially available aromatic thiols  when the reaction was carried out in 100 mM sodium phosphate buffer, pH 7.4, at a temperature between 0 °C and 25 °C, and using 2–3 equivalents of the sodium salt of the thiol, which was independently prepared by reaction of the thiol with one equivalent of sodium tert-butoxide. Therefore, under these reaction conditions, most of the assayed thiols led to the rapid and high-yield formation of the corresponding adduct, visible by the development of an intense yellow colour. This was particularly true for adducts I and V, whereas the formation of adduct VI was significantly less clean and lower yielding, most likely due to the high steric hindrance caused by the substitution of the two positions ortho to the thiolate group.It was found that the di-thiol adduct was essentially the only product (as shown by thin layer chromatography and V) was selected for the generation of antibodies because this compound is expected to possess good immunogenic properties due to the presence of two bromine atoms.It was also observed that the di-thiol adduct was predominant even when one equivalent of the thiolate was used, which indicates that, once a thiolate group reacted with patulin, the incorporation of the second thiolate group to the initially formed mono-thiol adduct is favoured over the addition of the first one to an intact patulin molecule. Moreover, the di-thiol adduct was the major product even when excess thiolate was employed if relatively short incubation times were used . These results were encouraging, opening up the possibility of attaining the goal of developing rapid point-of-need tests based on the derivatization of patulin with a thiolated compound. The adduct resulting from the reaction of patulin with 4-bromothiophenol . This is most likely due to the presence of the spacer arm in hapten V4, which probably favours a better display of the molecule to the surrounding medium, and thus a better interaction with the antibody may occur. More importantly, when the thiolate at 2 mg/mL in water was added (2% (v/v)) to patulin solutions in buffer, and the mixtures were assayed by competitive ELISA after incubation for 1 h at room temperature, the resulting inhibition curve overlapped with the curve that was obtained with the purified adduct V , although certainly outstanding for such a small compound, is still lower than desirable for the development of an immunoassay enabling the reliable determination of patulin at the more challenging regulated level (10 µg/kg). However, it does mean an encouraging proof-of-concept that the electrophilic reactivity of patulin can be harnessed to generate suitable antibodies for food immunodiagnostics.D,app = 35 nM). Although there is still room for improvement, this strategy opens new avenues for future developments on antibody-based methods for sensitive immunodetection of this challenging mycotoxin. Further work should address innovative ways of preparing more rigid and antigenic adducts of patulin that might allow the generation of antibodies with even superior binding properties, for example using aromatic 1,2-dithiols instead of monothiols. If this can be achieved, the long-standing goal of monitoring patulin contamination by widely established immunochemical methods in food immunodiagnostics, such as ELISA, immunostrips, and immunoaffinity columns, will become a reality.Three functionalized derivatives of patulin that conformed to commonly accepted rules in hapten design for antibody production were synthesized in the present study. Unfortunately, these conventional approaches did not lead to the production of antibodies that could recognize free patulin in solution. In addition to the small size of the molecule, the high electrophilic reactivity of patulin is most likely responsible for these negative results. This disappointing outcome prompted us to explore a disruptive approach consisting of exploiting the high reactivity of patulin towards nucleophiles. Hence, antibodies against the adduct resulting from the reaction between patulin and a thiol-containing compound, 4-bromothiophenol, were obtained. Remarkably, the affinities of the antibodies thus generated for the patulin adduct were in the nanomolar range -5-((4-bromophenyl)thio)-3-(((4-bromophenyl)thio)methylene)-6-hydroxy-4-oxohexanoic acid (adduct V). A solution of 4-bromobenzenethiol  and sodium tert-butoxide  in anhydrous MeOH was stirred at 50 °C for 1 h under nitrogen atmosphere. The solvent was eliminated under reduced pressure to afford sodium 4-bromobenzenethiolate as a white powder. A solution of patulin  in 8 mL of 100 mM sodium phosphate buffer (PB), pH 7.4, was cooled in an ice-water bath and a solution of the above sodium thiolate  in Mili-Q H2O (5 mL) was added dropwise. The reaction mixture turned an intense yellow color almost immediately, which indicates how quickly the reaction is taking place. After 10–20 min stirring at the same temperature, the reaction mixture was acidified with formic acid to pH 3–4 and extracted with EtOAc (3 × 20 mL). The combined organic layers were washed with brine (15 mL), dried over anhydrous MgSO4, concentrated in vacuo, and finally purified by silica gel flash chromatography, using CHCl3–MeOH (95:5) with 0.1% of HCO2H as eluent, to afford adduct V as a yellow oil . The spectroscopic data of adduct V were in complete agreement with those described previously by Metzler. IR vmax (cm−1) 3339 m, 1705 m, 1472 m, 1230w, 1112w, 1019 s, 814 m; 1H NMR  δ 7.62 , 7.50 , 7.49 , 7.43 , 4.30 , 4.00 , 3.78 , 3.77 , 3.71 ; 13C NMR  δ 194.4 (C-4), 172.7 (C-1), 148.8 (HC = C3), 138.0 (C-3), 136.9 , 133.3 , 133.0 , 132.8 , 132.0 (C-1′ Ph), 127.0 (C-1 Ph), 123.3 (C-4′ Ph), 122.5 (C-4 Ph), 62.3 (C-6), 54.2 (C-5), 38.2 (C-2); HRMS (ESI) m/z calcd for C19H17Br2O4S2 [M+H]+ 530.8930 (51.4%), 532.8909 (100%) and 534.8889 (48.6%), found 530.8910, 532.8889 and 534.8867.V. The most relevant data for each case are given below.The procedure followed for the reaction of patulin with the other aromatic thiols was similar to that described above for the preparation of adduct (Z)-6-Hydroxy-4-oxo-5-(p-tolylthio)-3-((p-tolylthio)methylene)hexanoic acid (Adduct I). This adduct , a yellow oil, was obtained from a solution of patulin  in 100 mM PB (12.8 mL) and a solution of sodium 4-methylbenzenethiolate  in Mili-Q H2O (6.4 mL) at rt. The EtOAc extract of the reaction mixture was purified by silica gel flash chromatography using CHCl3–MeOH (from 98:2 to 95:5) as eluent. IR vmax (cm−1) 3021 m, 1713 m, 1493 m, 1216 s; 1H NMR  δ 7.38 , 7.27 , 7.25 , 7.19 , 7.09 , 4.10 , 4.04 , 3.88 , 3.84 , 3.40 , 2.37 and 2.31 ; 13C NMR  δ 194.0 (C-4), 175.1 (C-1), 152.9 (HC = C3), 139.7 (C-4′ Ph), 138.8 (C-4 Ph), 135.5 , 133.8 (C-1′ Ph), 131.2 , 130.3 , 130.1 , 125.7 (C-1 Ph), 123.9 (C-3), 61.5 (C-6), 52.4 (C-5), 38.1 (C-2), 21.4 and 21.3 (2xMePh); HRMS calcd for C21H23O4S2 [M+H]+ 403.1032, found 403.1018.(Z)-5-thio)-3-thio)methylene)-6-hydroxy-4-oxohexanoic acid (Adduct II). This adduct , a yellow oil, was obtained from a solution of patulin ) in 100 mM PB (5.4 mL) and a solution of sodium 2,4-dimethylbenzenethiolate  in Mili-Q H2O (5.4 mL) at rt. The EtOAc extract of the reaction mixture was purified by silica gel flash chromatography using CHCl3–MeOH 95:5 as eluent. IR vmax (cm−1) 3354 m, 3009 m, 2920 m, 2855 m, 1701 m, 1647 m, 1559 m, 1217 m, 1051 m, 954 m, 812 m, 751 s; 1H NMR  δ 7.34 , 7.24 , 7.08–6.95 , 7.01 , 6.91 , 4.16 , 4.03 , 3.84 , 3.69 , 3.29 , 2.39, 2.35, 2.32 and 2.26 ; 13C NMR  δ 194.4 (C-4), 174.3 (C-1), 152.6 (HC=C3), 142.2 (C-2′ Ph), 139.2 (C-2 Ph), 137.6 (C-4′ Ph), 136.2 (C-4 Ph), 131.4 , 131.2 (C-6′ Ph), 129.9 (C-1′ Ph), 127.7 , 127.3 (C-5′ Ph), 125.8 (C-1 Ph), 123.3 (C-3), 61.7 (C-6), 52.2 (C-5), 21.1, 21.0, 20.9 and 20.8 (4xMePh); HRMS calcd for C23H27O4S2 [M+H]+ 431.1345, found 431.1346.(Z)-4,4'-((2-(Carboxymethyl)-5-hydroxy-3-oxopent-1-ene-1,4-diyl)bis(sulfanediyl)) dibenzoic acid (Adduct III). This adduct , a yellow oil, was obtained from a solution of patulin  in 100 mM PB (4.0 mL) and a solution of the 4-mercaptobenzoic acid  in Mili-Q H2O (4 mL) at rt. The EtOAc extract of the reaction mixture was purified by C18-reversed-phase flash chromatography using MeOH–H2O 7:3 as eluent. IR vmax (cm−1) 3060 m, 2361 m, 1698 s, 1596 s, 1421 m, 1281 m; 1H NMR  [only signals of major Z-isomer are given] δ 8.06 , 7.96 , 7.68 , 7.65 , 7.60 , 4.54 , 4.09 , 3.87 , 3.78 ; 13C NMR  [only signals of major Z-isomer are given] δ 194.8 (C-3′′), 172.6 (C-2′′′), 167.0 and 166.9 (C-1 and C-1′), 147.4 (C-1′′), 144.1 (C-4 Ph), 140.2 (C-4′ Ph), 133.1 (C-1 Ph), 132.3–130.2 , 130.6 (C-1′ Ph), 127.6 (C-2′′), 62.8 (C-5′′), 53.9 (C-4′′), 38.2 (C-1′′′); HRMS calcd for C21H19O8S2 [M+H]+ 463.0516, found 463.0516.(Z)-2,2'-(((2-(Carboxymethyl)-5-hydroxy-3-oxopent-1-ene-1,4-diyl)bis(sulfanediyl)) bis)diacetic acid (Adduct IV). This adduct , a yellow oil, was obtained from a solution of patulin  in 100 mM PB (5.8 mL) and a solution of 2-(4-mercaptophenyl)acetic acid  in a 20 mM aqueous solution of NaOH  at rt. The ethyl acetate extract of the reaction mixture was purified by silica gel flash chromatography using CHCl3-MeOH 95:5, containing a 0.1% of HCO2H, as eluent. IR vmax (cm−1) 3021w, 1711s, 1363 m, 1221 s, 1093w; 1H NMR  δ 7.51 , 7.49 , 7.45 , 7.39 , 7.30 , 4.26 , 4.00 , 3.79 and 3.71 , 3.77 , 3.68 and 3.64 ; 13C NMR  δ 194.6 (C-3′′), 172.8 (C-2′′′), 172.5 and 172.4 (C-1 and C-1′), 149.9 (C-1′′), 147.7 (C-4 Ph), 136.9, 136.5 and 136.1, 135.0 , 131.4 , 131.3 , 131.0 , 126.5 (C-2′′), 62.4 (C-5′′), 54.3 (C-4′′), 40.8 and 40.7 (C-2 and C-2′), 38.3 (C-1′′′); HRMS calcd for C23H23O8S2 [M+H]+ 491.0829, found 491.0810.(Z)-6-Hydroxy-5-(mesitylthio)-3-((mesitylthio)methylene)-4-oxohexanoic acid (Adduct VI). This adduct , a yellow oil, was obtained from a solution of patulin  in 100 mM PB (6.7 mL) and a solution of the sodium salt of 2,4,6-trimethylbenzenethiol  in 6.7 mL of Mili-Q H2O at rt. The EtOAc extract of the reaction mixture was purified by silica gel flash chromatography using CHCl3–MeOH 95:5 as eluent. As deduced from the analysis of the 1H NMR spectrum, this compound exists in solution as a mixture of two isomers, most probably rotamers around the C–S bonds. IR vmax (cm−1) 3330 m, 2922 m, 2855 m, 2100w, 1714s, 1671 m, 1619 m, 1602 m, 1457 m, 1399 m, 1375 m, 1164 s, 1034 m, 1012 m, 851 m, 754 s; 1H NMR  [only signal of the major rotamer are given] δ 7.02 , 6.97 , 6.83 , 4.17 , 3.97 , 3.75 , 3.27 and 3.05 , 2.50 , 2.38 , 2.26 and 2.24 ; HRMS calcd for C16H19O4S [M–Me3PhS]+ 307.0999, found 307.0998.V , EDC·HCl  and NHS  in CH2Cl2 (0.8 mL) was stirred at − 20 °C for 1 h. The solvent was eliminated under vacuum and the residue was dissolved in dry MeCN (0.4 mL). In a separate vial, β-alanine  and NaHCO3  were dissolved in Mili-Q H2O (0.4 mL), this solution was added to the former, and the mixture was stirred for 1 h at 0 °C and 1 h at rt. The reaction mixture was cooled in an ice bath, acidified with citric acid, and extracted with EtOAc (3 × 20 mL). The combined organic layers were washed with brine (15 mL), dried over anhydrous MgSO4, concentrated in vacuo, and purified by silica gel flash chromatography, using CHCl3-MeOH (95:5) as eluent, to afford hapten V4 as a light yellow oil . IR vmax (cm−1) 3298 m, 1653w, 1448 m, 1230w, 1112w, 1019 s, 611 m; 1H NMR  δ 7.61 , 7.49 , 7.42 , 7.42 , 4.52 , 4.00 , 3.76 , 3.64 , 3.55  3.43 , 2.50 ; 13C NMR  δ 194.8 (C-4′), 172.2 (C-1), 169.3 (C-1′), 147.9 (HC=C3), 138.2 (C-3), 136.6 , 133.2 , 133.0 , 132.8 , 128.1 (C-1′′ Ph), 125.8 (C-1 Ph), 123.0 (C-4′′ Ph), 122.4 (C-4 Ph), 62.4 (C-6′), 53.9 (C-5′), 40.3 (C-3), 36.2 (C-2′), 34.4 (C-2); HRMS (ESI) m/z calcd for C22H22Br2NO5S2 [M+H]+ 601.9301 (51.4%), 603.9280 (100%) and 605.9260 (48.6%), found 601.9298, 603.9274 and 605.9270.A mixture of adduct Preparation of 2,5-dioxopyrrolidin-1-yl (Z)-5-((4-bromophenyl)thio)-3-(((4-bromophenyl) thio)methylene)-6-hydroxy-4-oxohexanoate (V–NHS). Adduct V , NHS  and EDC∙HCl  were dissolved in of anhydrous CH2Cl2 (0.5 mL) and stirred for 1 h at − 20 °C under nitrogen. It was observed that if the formation of the active ester is carried out at higher temperatures, the OH at C-6 is eliminated, resulting in the formation of a terminal double bond between C-5 and C-6. For example, the active ester of the mentioned elimination product is formed mostly at room temperature. Upon completion, the reaction mixture was diluted with CH2Cl2 and washed successively with water and brine, and then dried over anhydrous MgSO4. Evaporation of the solvent under reduced pressure afforded compound V–NHS  as a brownish oil. 1H NMR  δ 7.54 , 7.43 , 7.41 , 7.37 , 7.26 , 4.13 , 4.06 , 4.05 , 3.87 , 3.76 , 2.85 2CO).Preparation of 2,5-dioxopyrrolidin-1-yl (Z)-3-(5-((4-bromophenyl)thio)-3-(((4-bromophenyl)thio)methylene)-6-hydroxy-4-oxohexanamido)propanoate (V4–NHS). Hapten V4 , NHS  and EDC∙HCl  were dissolved in anhydrous CH2Cl2 (0.5 mL) and stirred for 1 h at − 20 °C under nitrogen atmosphere. Upon completion, the reaction mixture was diluted with CH2Cl2 and washed successively with water and brine, and then dried over anhydrous MgSO4. Evaporation of the solvent under reduced pressure afforded compound V4–NHS  as a brownish oil. 1H NMR  δ 7.62 , 7.51 , 7.50 , 7.49 , 7.42 , 4.48 , 4.00 , 3.76 , 3.66 , 3.57 , 3.56 , 2.88 , 2.87 2CO).N,N-dimethylformamide (or MeCN) to a 15 mg/mL BSA or OVA solution in 100 mM phosphate buffer, pH 7.4, under gentle stirring overnight at room temperature. BSA–hapten immunizing conjugates were prepared by employing 30 , 40 (hapten V4) or 60 (adduct V) mol of activated hapten per mol of protein, whereas for OVA–hapten assay conjugates 10  or 15 (adduct V and hapten V4) equivalents were used. Conjugates were purified by size exclusion chromatography using three connected 5 mL Sephadex G-25 HiTrap desalting columns and using 100 mM phosphate buffer, pH 7.4, as eluent. BSA conjugates were filter-sterilized through 0.45 μm pore filters and stored at − 20 °C. Final hapten-to-protein molar ratios were determined by Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF/MS).Following activation, hapten coupling was carried out by dropwise adding a 50 mM solution of the active ester in Analysis of hapten bioconjugates was performed by mass spectrometry as follows:Sample preparation. 100 μL of protein conjugate (0.5‒1 mg/mL) were dialyzed against milliQ water and lyophilized. The sample was dissolved in MilliQ water to a theoretical final concentration of 1 mg/mL, and 1 μL was spotted onto the MALDI plate. After the droplet was air-dried at room temperature, 1 μL of matrix (10 mg/mL sinapinic acid (Bruker) in 70% MeCN, 0.1% Trifluoroacetic acid) was added and allowed to air-dry at room temperature.Mass spectrometry analysis. The sample was analyzed in a 5800 MALDI TOF/TOF (ABSciex) instrument in positive linear mode (1500 shots every position) in a mass range of 15,000‒100,000 m/z. Previously, the plate was calibrated with 1 μL of the TOF/TOF calibration mixture (ABSciex), in 13 positions. Every sample was calibrated by ‘close external calibration’ method with a BSA or OVA spectrum acquired in a close position. The analysis of the results was performed using the mMass program (http://www.mmass.org/).Experimental design was approved by the Bioethics Committee of the University of Valencia (procedure number 2019/VSC/PEA/0179). The study was carried out in compliance with the ARRIVE guidelines. Animal manipulation was performed in compliance with the European Directive 2010/63/EU and the Spanish laws and guidelines (RD1201/2005 and 32/2007) concerning the protection of animals used for scientific purposes. Female New Zealand white rabbits were immunized by subcutaneous injection with 300 μg of the corresponding BSA–hapten conjugate. The immunogen consisted of a 1:1 emulsion between the conjugate in PBS and Freund’s adjuvant (complete for the first inoculation and incomplete for subsequent boosts). Four injections were applied at three-week intervals, followed by animal exsanguination by intracardiac puncture ten days after the last injection. Blood samples were left to coagulate at 4 °C overnight and the serum was separated by centrifugation. Antibodies were precipitated twice with a saturated ammonium sulfate solution in water and stored at 4 °C.2SO4, and the absorbances at 450 nm were immediately read using a reference wavelength at 650 nm. The experimental values were fitted to a four-parameter logistic equation.Immunoassays were carried out in the indirect ELISA format. Microtiter plates were coated by adding 100 µL per well of the corresponding OVA–hapten conjugate in 50 mM carbonate-bicarbonate buffer, pH 9.6. After overnight incubation at room temperature, the plates were washed four times with a 150 mM NaCl solution. The competitive assay was carried out by adding 50 μL per well of the analyte in PBS plus 50 μL per well of antiserum solution in PBST (PBS containing 0.05% (v/v) Tween 20), and the plates were incubated one hour at room temperature. After plate washing, 100 μL per well of the enzyme-labeled secondary antibody (GAR–HRP (1/10000)) in PBST containing 10% (v/v) fetal bovine serum was added and incubated at room temperature for an additional hour. The plates were washed again and the signal was generated by adding 100 µL per well of TMB enzyme substrate solution. Following incubation during 10 min at room temperature, the reaction was stopped by adding 100 µL per well of 1 M HSupplementary Information.


Cancer cells escape macrophage phagocytosis by exploiting the CD47/SIRPα axis. We found that extensive membranous CD47 expression by cancer cells characterized 29/98 cases. SIRPα and CD68 were expressed by tumor-associated macrophages . A high CD68Mφ-score was linked with improved overall survival. High expression, however, of SIRPα by CD68+ TAMs was linked with CD47 expression by cancer cells, low TIL-score, and poor prognosis. A direct association of CD47 expression by cancer cells and the % FOXP3+ TILs was also noted. The CD47/SIRPα axis is a sound target for adjuvant immunotherapy policies, aiming to improve the cure rates in operable NSCLC.p = 0.005); and this was independent of the stage . In contrast, high SIRPα expression by CD68+ TAMs (SIRPα/CD68-ratio) was linked with CD47 expression by cancer cells, low TIL-score, and poor prognosis (p = 0.02). A direct association of CD47 expression by cancer cells and the % FOXP3+ TILs  was also noted. Conclusions: TAMs play an important role in the prognosis of operable NSCLC. As SIRPα+ macrophages adversely affect prognosis, it is suggested that the CD47/SIRPα axis is a sound target for adjuvant immunotherapy policies, aiming to improve the cure rates in operable NSCLC.Background: Cancer cells escape macrophage phagocytosis by expressing the CD47 integrin-associated protein that binds to the SIRPα ligand  expressed by macrophages. Immunotherapy targeting this pathway is under clinical development. Methods: We investigated the expression of CD47/SIRPα molecules in a series of 98 NSCLCs, in parallel with the infiltration of tumor stroma by CD68+ macrophages, tumor-infiltrating lymphocytes (TILs), and PD-L1/PD-1 molecules. Results: Extensive membranous CD47 expression by cancer cells characterized 29/98 cases. SIRPα and CD68 were expressed, to a varying extent, by tumor-associated macrophages . A high CD68Mφ-score in inner tumor areas was linked with improved overall survival ( The development of monoclonal antibodies targeting immune-checkpoint pathways opened the most promising current era of cancer immunotherapy. A large number of therapeutic antibodies have been approved for clinical applications, which at present target the PD-L1/PD-1 pathway and the CTLA4 inhibitory ligand of CD80/CD86 molecules. Both pathways aim to enhance the cytotoxic activity of T-cells . Cancer The expression patterns of CD47 in human malignancies have been investigated in a small number of published studies, mainly from Eastern Institutes. Association with aggressive clinical behavior and poor prognosis have been reported in breast , esophagIn the current study, we investigated the CD47/SIRPα axis in a series of NSCLCs by examining in parallel the immunohistochemical patterns of expression of CD47, SIRPα, and CD68 pan-macrophage marker. Their correlation with histopathological characteristics and the postoperative prognosis is reported. Moreover, we analyze the association of CD47/SIRPα with the PD-L1/PD-1 axis and with parameters related to tumor-infiltrating lymphocytes.In a normal lung, CD47 was expressed by the bronchial epithelium a, alveolp = 0.05). There was no association with histology type (p = 0.72). Out of 58 squamous cell carcinomas 21 (36.2%) were negative for CD47, 20 (34.5%) had medium expression and 17 (29.3%) had high expression. Out of 22 adenocarcinomas, 7 (31.8%) were negative, 7 (31.8%) had medium expression and 8 (36.4%) had high expression. Out of 18 undifferentiated tumors, 5  were negative, 9 (50%) had medium expression and 4 (22.2%) had high expression.CD47 was expressed in the membrane and cytoplasm of cancer cells c,d. The p = 0.83). Out of 33 tumors with a low proliferation index 11 (33.3%) were negative for CD47, 10 (30.3%) had medium expression and 12 (36.4%) had high expression. Out of 46 tumors with a medium proliferation index, 15 (32.6%) were negative, 19 (41.3%) had medium expression and 12 (26.1%) had high expression. Out of 19 tumors with a high proliferation index, 7 (36.8%) were negative, 7 (36.8%) had medium expression and 5 (26.4%) had high expression.Similarly, there was no association of CD47 expression with and MIB1 proliferation index  or inner tumors areas (p = 0.90).SIRPα and CD68 were not expressed by cancer cells but rather by tumor-associated macrophages  . The extp < 0.0001, r = 0.79). There was no statistical association between SIRPα-score, histology type, stage of disease, or MIB proliferation index. The mean SIRPαMφ-score in the invading front was 0.75, 0.95, and 0.72 in the squamous cell carcinomas, adenocarcinomas, and undifferentiated carcinomas, respectively (p > 0.17). The mean SIRPαMφ-score in inner tumor areas was 0.75, 0.81, and 0.66 in the squamous cell carcinomas, adenocarcinomas, and undifferentiated carcinomas, respectively (p > 0.38). The mean SIRPαMφ-score in the invading front was 0.78, 0.90 and 0.73 in stage 1, 2 and 3, respectively (p > 0.32). The mean SIRPαMφ-score in inner tumor areas was 0.69, 0.90 and 0.73 in stage 1, 2 and 3, respectively (p > 0.11). The mean SIRPαMφ-score in the invading front was 0.69, 0.76, and 0.1.05 in tumors with low, medium, and high MIB1 proliferation index, respectively. Although tumors with a high proliferation index had a higher SIRPαMφ-score, the statistical difference was marginal (p > 0.06). The mean SIRPαMφ-score in inner tumor areas was 0.69, 0.71, and 0.94 in tumors with low, medium, and high MIB1 proliferation index, respectively (p > 0.12).Linear regression analysis revealed a strong direct association of SIRPαΜφ-score between tumor invading front and inner tumor areas . There was no association of the CD68Mφ-score with the histology, stage. The mean CD68Mφ-score in the invading front was 1.8, 2.1, and 2.2 in the squamous cell carcinomas, adenocarcinomas, and undifferentiated carcinomas, respectively (p > 0.15). The mean CD68Mφ-score in inner tumor areas was 1.8, 1.8, and 2.1 in the squamous cell carcinomas, adenocarcinomas, and undifferentiated carcinomas, respectively (p > 0.26). The mean CD68Mφ-score in the invading front was 2.1, 1.8 and 2.0 in stage 1, 2 and 3, respectively (p > 0.12). The mean CD68Mφ-score in inner tumor areas was 1.9, 1.7, and 1.9 in stages 1, 2 and 3, respectively (p > 0.27). MIB1 proliferation index was significantly higher in tumors with a high CD68Mφ-score, especially in inner tumor areas .In addition, the same analysis revealed a strong direct association of CD68Mφ-score between tumor invading front and inner tumor areas (p > 0.95) , whether calculated in the invading tumor front or inner tumor areas ( > 0.95) b.p = 0.31). A strong association of high CD68Mφ-score in inner tumor areas with better overall survival was noted (p = 0.005); p = 0.37; stage II/III: p = 0.05)Furthermore, survival analysis of disease-specific survival  did not reveal any significant association with CD68Mφ-score when calculated in the invading tumor front  together with the stage .In a bivariate model of regression analysis of death events, a high CD68Mφ-score was an independent prognostic variable , the ratio differed in 30/98 cases.The density of Mφ expressing SIRPα among CD68+ Μφ was scored by dividing the SIRPαMφ-score bythe CD68Mφ-score. This SIRPα/CD68-ratio ranged from 0 to 1, and its distribution is shown in p = 0.02; p = 0.74; A ratio of 0–0.3 was considered low, while 0.5–1 was high. Kaplan–Meir survival analysis of disease-specific survival  showed a direct association of high SIRPα/CD68-ratio in inner tumor areas with poor prognosis . Thus, 54/98 NSCLCs expressing CD47 also contained SIRPα+ infiltrating Mφs. Out of 29 NSCLCs with a high expression of CD47, 25 were also infiltrated by SIRPα expressing Μφs. A direct association was also noted with the SIRPα/CD68-ratio  and with the FIL-score  was noted in linear regression analysis  was recorded in one-third of tumors. Although no association was found with histopathological parameters, dense TAM infiltration was linked with a high MIB1 proliferation index. However, survival analysis showed that dense TAM presence was significantly related to a better postoperative outcome. This finding suggests that macrophage infiltration and related innate immunity does, indeed, exert anti-tumor activity in a fraction of tumors. Of interest, the survival benefit was more prominent in stage II/III disease. As for most patients with advanced local disease, death occurs from metastasis, our findings support the idea that recognition of tumors by macrophages may have contributed to the eradication of micro-metastatic disease till the time of the operation. Immunotherapeutic approaches that would enhance macrophage activity may, therefore, prove of value in the improvement of cure rates offered by surgery, in patients with compromised macrophage innate immunity.Nonetheless, the CD68 marker does not discriminate between M1 or M2 macrophage phenotypes or immune checkpoint molecules exploited by cancer cells to block macrophage activity. In the current study, we focused on this latter issue, by investigating the CD47/SIRPα inhibitory checkpoint axis. The CD47/SIRPα axis is an important pathway in normal physiology that blocks the phagocytic activity of macrophages. SIRPα is a glycoprotein abundant in neurons and the myeloid lineage of hemopoietic cells, including macrophages. Expression of the CD47 integrin by most of the normal cells blocks the phagocytic activity of macrophages by binding to their extracellular domain of SIRPα. Mature red blood cells, for example, lose their CD47 molecules, becoming a target for their elimination from the bloodstream by macrophages . AdoptinThere are only a few studies published on the clinicopathological role of CD47 in NSCLC. Arrieta et al. examined tumor biopsies from 169 NSCLCs, showing expression of CD47 in 84% of tumors, and although there was no overall association with prognosis, CD47 defined better prognosis in patients with EGFR mutations . Yang etIn our study, analysis of NSCLC patients showed that membrane CD47 overexpression by cancer cells was noted in two-thirds of tumor samples, where one-third expressed CD47 in more than 50% of cancer cells. This finding shows that, indeed, the majority of NSCLCs can mask themselves against the macrophages. In addition, CD47 overexpression by cancer cells was directly related to SIRPα expression by TAMs and not with CD68+ TAMs, showing that an interplay between CD47 expressing cancer cells and the SIRPα+ Μφ exists in NSCLC. This hypothesis was further substantiated in the survival analysis. Although dense CD68+ TAM presence was linked with good prognosis, expression of SIRPα by these TAMs (high SIRPα/CD68-ratio) was strongly linked with poor postoperative prognosis. Eventually, SIRPα emerges as a potent marker to identify immunosuppressive type M2 macrophages. As the CD47/SIRPα cancer cell/Mφ axis is commonly expressed in NSCLC a therapeutic blockage could prove useful as adjuvant or neo-adjuvant immunotherapy in operable NSCLC .Analysis of TAMs and CD47/SIRPα expression patterns did not reveal any association with the expression of PD-L1/PD-1, another important immune checkpoint pathway related to cytotoxic T-cell activity in NSCLC. The direct association of CD47 expression, however, with poor stroma infiltration by TILs and, most importantly, with dense FOXP3+ TIL presence, is an additional interesting observation linking macrophage and T-cell immunosuppressive pathways in a subset of NSCLCs. This is in accordance with a previous study in gastric cancer, where CD47 expression was linked with a lower CD8/FOXP3 ratio . Tseng eNinety-eight sequential tissue samples of surgically resected NSCLC were retrieved from the archives of the Department of Pathology, Democritus University of Thrace. Tissue sections were selected to contain the invading tumor front of the tumor, so these also contained adjacent normal lung tissue. All patients were treated with surgery alone. The median age of patients was 68 years (range 32–82). According to the UICC staging system, 46/98 patients were of stage I, 22/98 of stage II, and 30/98 of stage III. Regarding histology, 58 cases were of the squamous type, 22 cases were adenocarcinomas, and 18 were undifferentiated carcinomas. Out of 58 squamous cell carcinomas, 30 were of grade 1, 15 of grade 2, and 13 of grade 3. Out of 22 adenocarcinomas, 7 were of grade 1, 10 of grade 2, and 5 of grade 3. The median follow-up of patients was 46 months, ranging from 26–112 months.The study was conducted according to the criteria set by the declaration of Helsinki, after approval from the Scientific and Ethics Committee of the University Hospital of Alexandroupolis .Formalin-fixed paraffin-embedded tissue sections of 3 μm thick were cut and placed on poly-L-lysine slides. For CD47 detection, we used the primary mouse monoclonal ab260418 , with 60 min incubation, at dilution 1/200. For the detection of SIRPα we used the primary rabbit monoclonal ab260039 antibody , with 60 min incubation, at dilution 1/250. Immunohistochemistry for CD68 was performed using the mouse monoclonal KP1 clone (Immunologic) at dilution 1/300, with 60 min incubation. The heat-induced epitope retrieval process was performed in a microwave oven using Dako EnVision FLEX Target Retrieval Solution (pH 9.0). After washing the specimen twice for 6 min each time, the slides were incubated with the primary antibodies. Following washing with PBS, endogenous peroxidase was quenched with EnVision Flex Peroxidase Block  for 10 min. Non-specific binding was blocked in EnVision Flex mouse or rabbit Linker, as appropriate, for 15 min (DAKO) and washed in PBS. Subsequently, slides were incubated with the secondary antibody  for 30 min. The color was developed by 5 min incubation with EnVision Flex Chromogen (DAKO), and sections were counterstained weakly with hematoxylin. In every staining run, the primary antibody was replaced by normal species-specific immunoglobulin-G from at least one section to provide a negative control.Assessment of the expression of CD47 was performed at ×20 magnification. The percentage of cancer cells with membrane expression was recorded in all available optical fields (o.f.). The mean value was used to score each tissue section. The expression of CD47 by stroma fibroblasts was also quantified. The number of ×20 o.f. showing strong expression divided by the total number of ×20 o.f. was used to score the extent of CD47 expression in the stroma for each case.The number of macrophages (Mφ) stained for SIRPα and CD68 was recorded in all ×40 magnification o.f., separately in the invading tumor front and the inner tumor stroma. As invading tumor front is defined the ×200 optical fields that contain the tumor are located in direct proximity to the normal lung tissue. All other optical fields, moving to central tumor areas, are considered inner tumor areas. The final grouping of cases according to the Μφ density (SIRPαΜφ-score and CD68Μφ-score) was performed as follows: Score 0: lack of expression, Score 1: less than 20 Mφ/o.f., Score 2: 20–50 Mφ/o.f. and Score 3: >50 Mφ/o.f. Subsequently, we calculated the SIRPα/CD68-ratio (for the invading front and inner tumor areas) by dividing the SIRPαΜφ-score/CD68Μφ-score. This ratio reflects the percentage of tumor-associated macrophages (TAMs) that express SIRPα.A sequential immunofluorescence staining protocol in two steps was applied on cancer tissue sections from 5 patients with extensive CD68+ and SIRPα+ and 5 tissue sections with extensive CD68+ but low SIRPα+ macrophage stroma infiltration documented in immunohistochemistry single staining. The anti-CD68 and anti-SIRPα antibodies were used at concentrations and conditions used in immunohistochemistry. Details on the protocol applied have been previously published by our group . The antThe quantification of tumor-infiltrating lymphocytes TIL-density in the tumor stroma has been previously described ,26. TILsWe further assessed the expression of PD-L1 by cancer cells and of PD-1 and FOXP3 in tumor-infiltrating lymphocytes (TILs), using the rabbit monoclonal anti-PD-L1 antibody clone CAL10 , the mouse monoclonal clone NAT105  and an ‘in house’ undiluted hybridoma supernatant from the well-validated anti-FOXP3 murine monoclonal antibody 236A/E7, respectively, as previously reported .For PD-L1, the percentage of cancer cells with strong membrane (with or without cytoplasmic) expression was recorded in all ×200 o.f., and the mean value was calculated to score each case. Cases were grouped into three categories thus negative, low (expression in 1–9% of the cancer cells), and high (expression in >10% of the cancer cells).PIL-score and the FIL-score were, therefore, assessed as the product of “TIL-score” ×“% PD-1 or FOXP3+ TILs”.The percentage of PD-1 and of FOXP3 expressing TILs among the total amount of eosin-stained TILs present in the tumor stroma was assessed in all ×40 o.f., and the mean score was calculated for each case. As this score provides the % of TILs expressing PD-1 or FOXP3, and does not reveal the extent of PD-1 and FOXP3 lymphocytic infiltration in the tissue, which also depends upon the extent of TIL presence. The p-value of <0.05 was considered for significance.For statistical analysis and graph presentation, we used the GraphPad Prism 7.0 software. The chi-square or Fisher’s exact t-test was used to compare categorical variables. The Pearson’s correlation analysis test was used to assess relations between continuous variables. The overall disease-specific survival was assessed with the Kaplan–Meier method. A Cox proportional hazard model was used for multivariate analysis of death events. A In conclusion, TAMs play an important role in the control of metastasis and the prognosis of operable NSCLC. CD47/SIRPα up-regulation is a common event in NSCLCs, and SIRPα expression by macrophages is linked with poor prognosis. Whether CD47/SIRPα is a critical immune checkpoint target for the development of adjuvant immunotherapy policies, aiming to improve the cure rates in operable NSCLC should be investigated in clinical studies. Further studies on M1/M2 phenotypic prevalence in NSCLC are ongoing, anticipating a prognostic and therapy guiding classification of tumors according to the patient macrophage innate immunity status.


Procambarus clarkii) shell after cooking. In this study, two red-change-related pigment proteins with molecular weights of approximately 170 and 43 kDa—denoted as F1 and F2, respectively—were purified by ammonium sulphate salting-out and size exclusion chromatography. F1 and F2 entirely comprised homomultimeric protein complexes composed of 21 kDa subunits. LC-MS/MS analysis showed that the 21 kDa protein subunit belonged to the crustacyanin family, named P. clarkii crustacyanin A2 (PcCRA2). The full-length cDNA of PcCRA2 was cloned, which encoded 190 amino acid residues and was highly homologous (91.58%) with Cherax quadricarinatus crustacyanin A. The predicted 3D structure showed that PcCRA2 had a β-barrel structure for pigment encapsulation. The colour change of F1 was first detected at 40 °C, and the red change occurred upon heating above 60 °C. Additionally, with increasing temperature, its β-sheet content increased, and its α-helix content reduced. Correlation analysis showed that the redness value of F1 was significantly related to the heating temperature and the β-sheet content.Pigment proteins play a vital role in the red colour change of the red swamp crayfish ( Eriocher sinesis) draws consumers’ attention and acquires higher market prices than dark orange or yellow crabs 2SO4)  and then stirred overnight at 4 °C. After centrifugation at 16,000× g at 4 °C for 20 min, each resulting supernatant was collected separately, and the resulting precipitate was dissolved in sodium phosphate buffer . The total protein content of each supernatant and precipitate was measured using the Bradford protein assay kit , and bovine serum albumin was used as a reference. The colour change of each supernatant and precipitate was measured after heating at 100 °C for 10 min.The crude pigment proteins . The resulting primary pigment proteins were further purified by size exclusion chromatography (SEC) using an ÄKTA purifier high-performance liquid chromatography system ). In short, the column was equilibrated and eluted with NaCl (0.15 M) and sodium phosphate buffer (20 mM) to adjust the pH to 7.0. The flow rate was 1.0 mL·min−1. Fractions were collected according to a real-time peak value at 280 nm, the concentration was adjusted to 1 mg·mL−1, and the colour change was observed after heating at 100 °C for 10 min. The purity of the eluted fractions was determined by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE).The precipitate obtained by the [NH−1, 20 μL) was loaded onto a TSK-gel G3000SWxl column , eluted with sodium phosphate buffer  at a flow rate of 0.7 mL·min−1, and monitored at UV 280 nm. In addition, protein standard mix (including thyroglobulin (670 kDa), γ-globulins from bovine blood (150 kDa), albumin chicken egg grade VI (44.3 kDa), ribonuclease A (13.7 kDa), and p-aminobenzoic acid (PABA), Sigma-Aldrich, St. Louis, MO, USA) were used to calibrate the column.The molecular weight of native purified pigment proteins was assessed using size exclusion chromatography (SEC). The sample , and the lightness parameter (L*), redness , and yellowness  were recorded. The total colour difference (dE) was calculated according to Equation (1):g for 10 min at 4 °C. The supernatants and precipitates were collected and analysed using SDS-PAGE in a 5% stacking gel and a 12% separating gel. Before electrophoresis, the precipitate was suspended in 0.5 mL of deionised water and homogenised by a vortex mixer at 2800 rpm . The gels were stained using a Coomassie blue staining solution  and scanned using a gel imaging system .The above thermally treated primary pigment-protein samples were centrifuged at 10,000× −1; Promega, Madison, WI, USA). The digested solutions were subjected to peptide sequencing analysis by LC-MS/MS with a Q Exactive Orbitrap HF mass spectrometer with a nanoelectrospray ionization source  and an LC-20AD nano high-performance liquid chromatography (HPLC) system equipped with a MonoCap C18 trap column .The target protein bands in the stained SDS-PAGE gels were manually excised and de-stained by acetonitrile (50%), then treated with dithiothreitol (10 mM) and iodoacetamide (55 mM), and finally digested overnight at 37 °C with sequencing-grade trypsin  and B (95% acetonitrile containing 0.1% formic acid). Samples were loaded by mobile phase A at a flow rate of 8 μL·minThe resulting nanoflow liquid chromatography eluate was directly subjected to nanoelectrospray ionization, followed by mass spectrometry in the positive-ion mode. The electrospray voltage and capillary temperature were 1.6 kV and 250 °C, respectively. The MS scan was acquired within an m/z range of 350 to 2000 in the Orbitrap at a resolution of 70,000. The MS/MS scans were detected in a high-energy collisional dissociation operating mode with 27.0% normalised collision energy. Identical ions that were detected more than two times within 15 s were dynamically excluded.The collected MS/MS profiles were analysed with Proteome Discoverer software  and searched for in the National Center for Biotechnology Information (NCBI) protein database using the following search parameters: fixed modifications, carbamidomethyl (C); variable modifications, oxidation (M); enzyme, trypsin; maximum missed cleavages, 2; peptide mass tolerance, 20 ppm; fragment mass tolerance, 0.6 Da; mass values, monoisotopic.P. clarkii, and DNA quality was assessed by electrophoresis on 1.0% agarose gel. The DNA sequence of PcCRA2 was amplified by degenerate PCR. Based on the highly conserved amino acid sequences MFTT(L/V)(V/I)AA and TAECVYRA among C. quadricarinatus crustacyanin A (GenBank: ALC79588.1) and P. vannamei crustacyanin A2 (GenBank: XP_027238673.1), two degenerate primers were designated as follows: A2-Fw, 5′-ATGTTTAC(C/A)ACA(C/G)TC(G/A)T(C/T)GCTGCT-3′ and A2-Rev, 5′- TTAAGCTCTGTAGAC(G/A)CA(CT)TC(A/G)GCCG-3′. Degenerate PCR for amplification of A2 used the genomic DNA of P. clarkii as the template. The PCR product was purified, ligated to T-Vector pMD19, and sequenced. Total RNA was extracted from the muscle of P. clarkii using Trizol column total RNA extraction kit . To confirm the introns and obtain the full-length complementary DNA (cDNA) of PcCRA2, reverse transcription PCR was performed using the primers CA2-Fw (5′-ATGTTTACCACTCTAATCGCT-3′) and CA2-Rev (5′- TTAAGCTCTGTAGACGCACTC- 3′).Total genomic DNA was isolated from the muscle of −1) was treated at different temperatures  for 10 min, filtered through a 0.22 µm syringe filter, and scanned three times at 1 nm·s−1. The spectral resolution was 0.5 nm, time per point 0.5 s, bandwidth 1.0 nm, and measured temperature 25 °C. The CD intensity is expressed as the mean molar ellipticity (θ) (deg·cm2·dmol−1). The secondary structure data was calculated using CDNN version 2.1 software.CD spectra were determined in the range of 190–250 nm by Chirascan circular dichroism spectrophotometry  with a quartz cuvette path length of 0.05 cm. The F1 sample . The self-optimised prediction method with alignment (SOPMA) online software was used to predict secondary structure. Homologous models were generated on the Swiss-model online server. PyMOL was used to analyse the obtained models with the PcCRA2 model from H. gammarus chosen as a template (Protein Data Bank code: 1gka). The quality of the model PcCRA2 was assessed by MolProbity and QMEAN server. Finally, the PcCRA2 and astaxanthin were docked using AutoDock Vina 4.0.The cloned sequences were first analysed via the NCBI’s online basic local alignment search tool (BLAST). Then, amino acid sequences of PcCRA2 were analysed by ProtParam. Next, the sequences were analysed for identity using DNAman Version 6 . The signal peptide was predicted by the Signal program  software  and are expressed as mean ± standard deviation (SD).4)2SO4 increases, different undenatured proteins precipitate from crude extracts and can be recovered. 4)2SO4 saturation of 10% and 20%, the total protein content of the supernatant decreased, but that of the recovered precipitate increased to 0.36 and 0.44 mg·g−1, respectively. After the heat treatment at 100 °C for 10 min, the supernatant turned colour from blue to red; however, there was no significant colour change in the precipitate. These results suggested that partially interfering proteins were removed by 20% (NH4)2SO4 saturation. When the (NH4)2SO4 saturation increased from 20% to 60%, the total protein content and total colour difference (dE) of the supernatant gradually decreased from 1.09 to 0.41 mg·g−1 and from 39.82 to 0.37, respectively. In the 60–80% (NH4)2SO4 saturation range, the dE of the supernatant remained almost constant at approximately 0.37, with no noticeable colour transition occurring upon heating at 100 °C for 10 min. This indicated that the remaining protein in the supernatant was not associated with red colour change. Therefore, an (NH4)2SO4 saturation of 20–60% was used to precipitate potential red-change-related primary pigment proteins.Ammonium sulphate precipitation is a classic and widely used method employed to preliminarily purify proteins owing to its low cost, low toxicity to protein, and convenient operation ,24. As tTo further confirm the red colour change of the primary pigment proteins, the primary pigment proteins were heated at 25 to 100 °C for 10 min . No signP. clarkii [P. monodon [P. vannamei and M. japonicus [Carcinus maenas (38.2 kDa), Asellus aquaticus L. (20 kDa), and Eulimnogammarus cyaneus (15 and 25 kDa) [Three fractions  were isolated from the primary pigment proteins by SEC. The content of F1 was markedly higher than that of F2 and F3 a. After  clarkii . However clarkii . This po clarkii . Two maj monodon . Additio monodon . A 75 kDaponicus . Similar 25 kDa) ,30,31.SEC of F1 and F2 revealed a prominent single peak for each, at retention times of 13.02 and 15.95 min, respectively see . The corp < 0.05). No known proteins with sequences similar to those of the 21 kDa subunits of F1 and F2 from P. clarkii were found, but crustacyanins from several shrimp species with scores of greater than 89 were discovered. Among these, C. quadricarinatus crustacyanin A was listed in the 21 kDa subunit of F1 and F2 with scores of 821 and 445, respectively (P. clarkii crustacyanin A2 (PcCRA2).Peptide sequences of the 21 kDa subunit of F1 and F2 were obtained by Q Exactive LC-MS/MS and compared with data in the NCBI database, respectively. A candidate protein was considered reliable provided the score was greater than 89 . The signal peptide region was present in the amino acid sequence between 1 and 16. The nucleotide sequence data were submitted to the GenBank database under accession numbers MW727506 and MW727507 see .C. quadricarinatus crustacyanin A (Accession: ALC79588.1), 87.93% similar to that of H.gammarus crustacyanin A2 subunit (Accession: P80007.1), and 76.32% similar to that of the P. vannamei crustacyanin A2 subunit-like isoform X1 (Accession: XP_027238673.1). The amino acid sequence of PcCRA2 contained three regions that are typical of the lipocalins family, including SCR-1 , SCR-2 (T-D-Y), and SCR-3 (R) [A multiple-protein sequence alignment revealed that PcCRA2 was highly homologous to the crustacyanins from other crustacean species . The amiH. gammarus crustacyanin A2 subunit as a template (PDA code: 1gka). The Ramachandran plot obtained from MolProbity analysis showed that 93.02% of the residues were located in the most favoured regions, whereas only 0.58% of the residues were in prohibited regions (data not shown). This suggested that the modelled structure of PcCRA2 was sufficiently accurate. Furthermore, the normalised QMEAN4 Z-score for the model PcCRA2 was found to be 0.92, which was in the range of the defined limits of the good quality model. The secondary structure prediction of PcCRA2 comprised random coils (42.63%), α-helixes (27.37%), β-sheets (23.68%), and a few β-turn (6.32%), as determined by SOPMA software. On this basis, the tertiary structure of PcCRA2 was modelled on the Swiss-model server using the existing crystal structures of −1) after the heat treatment at different temperatures for 10 min. With the temperature increasing from 25 to 100 °C, the lightness (L*), redness (a*), and yellowness (b*) gradually increased from 59.50 to 74.40, −12.57 to 26.03, and −25.67 to 25.70, respectively. The initiation of the colour change of F1 was detected at 40 °C, whereas the visible colour change occurred at 60 °C (p < 0.05). The red change was induced upon heating above 60 °C. The trend was consistent with the results of the colour change of the primary pigment proteins during heating.H. gammarus became red at 60 °C, but visible red precipitation required a higher temperature [P. clarkii carapace was closely related to temperature, which was in agreement with the findings of Pan et al., who reported no significant colour change in the pigment protein of P. vannamei at 30 to 60 °C and a gradual red colour change from 60 °C that was complete above 80 °C [Jasus lalandii started to change at 45 °C and turned orange at 65 °C and red at 85 °C [Britton et al. identified that pigment protein derived from perature . In the ve 80 °C . Timme eat 85 °C .θ) of the negative peak at 205–215 nm tended to increase , with the effect being more significant at above 60 °C. Additionally, the intensity of the strong positive peak near 195 nm remained unchanged below 60 °C, then gradually decreased with increasing temperature. These changes in the CD spectra indicated that the β-sheet content increased with increasing temperature, suggesting that the structure of the complex gradually unfolded.As can be seen in 2 = 0.889, R2 = 0.992, and R2 = 0.930, respectively).*; r = 0.962, p < 0.01). Additionally, the heating temperature was positively correlated with the β-sheet content  and negatively correlated with the content of α-helices, β-turns, and random coils .The correlation analysis of the heating temperature, secondary structural elements, and colour parameters are shown in C. quadricarinatus crustacyanin A and H.gammarus crustacyanin A2 and was denoted as P. clarkii crustacyanin A2. Prediction of the tertiary structure and molecular docking technology revealed several key amino acid residues  played an important role in combining with astaxanthin. When heated above 60 °C, F1 exhibited a visible red colour shift, and its β-sheet content increased with increasing temperature while its α-helix content decreased. Furthermore, the correlation analysis showed that the redness value (a*) of F1 was highly correlated with the heating temperature and the β-sheet content. These findings contribute to a better understanding of the mechanism of the red shift of pigment proteins in crustaceans.In this study, two novel red-change-related pigment proteins of 170 kDa (F1) and 43 kDa (F2) were purified. F1 and F2 were octamer and dimer proteins composed of eight and two 21 kDa subunits, respectively, which all belonged to the crustacyanin family. PCR analysis results showed that the 21 kDa protein subunit was highly similar to


Alphaproteobacteria isolate W30 from a historically polluted river in China through an HN-AD microbes screening process, which we identified as Pannonibacter sp. A potential HN-AD pathway for W30 was proposed based on N conversion analyses and the successful amplification of the entire denitrification gene series. The isolate exhibited high efficiency of aerobic inorganic nitrogen transformation, which accounted for 97.11% of NH4+-N, 100% of NO3−-N, and 99.98% of NO2−-N removal with a maximum linear rate of 10.21 mg/L/h, 10.46 mg/L/h, and 10.77 mg/L/h, respectively. Assimilation rather than denitrification was the main mechanism for the environmental nitrogen depletion mediated by W30. The effect of environmental constraints on aerobic NO3−-N removal were characterized, following a membrane bioreactor effluent test under an oxic condition. Compared to known Alphaproteobacterial HN-AD microbes, we showed that Pannonibacter sp. W30 could deplete nitrogen with no NO2−-N or NO3−-N accumulation in the HN-AD process. Therefore, the application of Pannonibacter sp. W30 has the potential for developing a felicitous HN-AD technology to treat N-laden wastewater at the full-scale level.The discovery of heterotrophic nitrification-aerobic denitrification (HN-AD) microorganisms has opened a new window for wastewater treatment. The underlying mechanism of HN-AD, however, is not fully understood because of the phylogenetic diversity of HN-AD microbes. The isolation and characterization of new HN-AD microorganisms are encouraging for furthering the understanding of this process. In this study, we found an  Alterna4+-N → NO2−-N → NO3−-N) compromise to low efficiency and incomplete N removal [Heavy organic and inorganic nitrogen (N) pollution in water bodies is a burgeoning environmental issue worldwide ,4,5,6. T removal . The dev removal .4+-N to NO3−-N and NO2−-N, and use O2, NO3−-N, and NO2−-N as the electron acceptors [Fueled by organic carbons, microbes in HN-AD processes can simultaneously transform NHcceptors ,9. Howevcceptors . Therefocceptors .Paracoccus denitrificans [Rhodococcus sp. [Pseudomonas sp. [Bacillus sp. [Alcaligenes sp. [Halomonas sp. [Alphaproteobacteria. Since most Alphaproteobacteria are oligotrophs that may adapt to nutrient variation in the environment, these microbes may apply novel mechanisms to tackle the low C/N ratio issue in HN-AD [HN-AD microbes are ubiquitous and can prevail in activated sludge ,13, aquarificans , Rhodococcus sp. , Pseudomonas sp. ,19,20, Bllus sp. , Alcaligenes sp. , and Halonas sp. . Among tin HN-AD .Pannonibacter sp., which belongs to Alphaproteobacteria. Its HN-AD capability was investigated using NH4+-N, NO3−-N, and NO2−-N as sole nitrogen sources, respectively. Based on N transformation assay and functional genes amplification, we suggest that isolate W30 is capable of carrying out nitrification and denitrification pathways. Since aerobic denitrification is the critical step for HN-AD, the species-specific N removal constraints were investigated. These factors were C/N ratio, carbon resource, dissolved oxygen, initial pH, temperature, and inoculation volume. Subsequently, the feasibility of isolate W30 in treating high N content wastewater at the full-scale level was tested using the effluent of a pilot membrane bioreactor (MBR). Our results suggest that (1) Pannonibacter sp. W30 is able to conduct HN-AD; (2) oxygen, however, is still a limiting factor affecting aerobic denitrification mediated by W30; (3) Pannonibacter sp. W30 adapts to a broad range of C/N ratios in wastewaters.In this study, we screened a historically eutrophic domestic river in Beijing, China, for isolating aerobic nitrogen removal bacteria. Isolate W30 was identified as 4)2SO4, 0.36 g/L KNO3, 4.0 g/L sodium citrate, and 0.05% (ratio of volume) of the trace element solution which was composed of 6.5 g/L K2HPO4·3H2O, 2.5 g/L MgSO4·7H2O, 2.5 g/L NaCl, 0.05 g/L FeSO4·7H2O, and 0.04 g/L MnSO4·H2O. The final pH of the EM was adjusted to 7.0.Enrichment medium (EM): The EM was composed of 0.5 g/L  of trace element solution which included 50.0 g/L EDTA-Na2, 2.2 g/L ZnSO4, 5.5 g/L CaCl2, 5.06 g/L MnCl2·4H2O, 5.0 g/L FeSO4·7H2O, 1.57 g/L CuSO4·5H2O, and 1.61 g/L CoCl2·6H2O. The final pH of the DM was adjusted to 7.0.Denitrifying medium (DM): The DM was composed of 0.36 g/L KNO3, 8.5 g/L sodium citrate, 1.0 g/L L-asparagine, 1.0 g/L KH2PO4, 1.0 g/L MgSO4·7H2O, 0.2 g/L CaCl2·6H2O, 0.05 g/L FeCl3·6H2O, and 0.1% (volume ratio) of the 1% (ratio of weight/volume) alcoholic dissolved bromothymol blue (BTB). The final pH of the DM was adjusted to 7.0. The solid GN was made by adding 2% of agar powder to the liquid GN.Screen medium (GN): The GN for the denitrifying bacteria was composed of 1.0 g/L KNOLuria-Bertani broth medium (LB): The LB broth liquid medium consisted of 10 g/L tryptone, 10 g/L yeast extract and 5 g/L NaCl, and 2% (g/L) of agar powder was added to LB broth liquid medium when solid plate medium was needed.6H5Na3O7, 0.24 g of (NH4)2SO4, 50 mL of HNM trace elements solution. The HNM trace elements solution contained (per liter): 5 g of K2HPO4 or 6.5 g of K2HPO4·3H2O, 2.5 g of MgSO4·7H2O and NaCl, 0.05 g of FeSO4·7H2O and MnSO4·4H2O [HNM medium consisted of the following components (per liter): 2.87 g of CSO4·4H2O .6H5Na3O7, 0.36 g of KNO3 (DM1) or 0.25 g of NaNO2 (DM2), 0.2 g of MgSO4·7H2O, 1.5 g of KH2PO4, 10.55 g of Na2HPO4·12H2O or 5.3 g of Na2HPO4·2H2O, 2 mL of DM trace elements solution. The DM trace elements solution contained (per liter): 50 g of EDTA-Na2, 2.2 g of ZnSO4·7H2O, 5.5 g of CaCl2, 5.06 g of MnCl2·4H2O, 5.0 g of FeSO4, 1.57 g of CuSO4·5H2O, and 1.6 g of CoCl2·6H2O.DM medium consisted of the following components (per liter): 2.87 g of CAll media were sterilized for 30 min at 0.11–0.15 MPa and 121 °C.−1) with sterilized distilled H2O and spread on solid screen medium with the pH indicator bromothymol blue at 30 °C. After cell colonies formed, the blue ones primitively confirmed as aerobic denitrifiers were picked up and incubated individually in liquid screen medium at 30 °C with shaking (150 rpm). Luria-Bertani (LB) broth medium was used to preserve the isolates at 4 °C.The method for isolating HN-AD microbes has been described previously . Briefly2 critical point dryer  and coated with gold . Cell morphology was observed using a scanning electron microscope  at an accelerating voltage of 15 kV. The 16S rRNA gene of W30 was amplified using the primer pair 27F/1492R [https://blast.ncbi.nlm.nih.gov/Blast.cgi/ (accessed on 1 November 2021). The information for denitrification genes was collected from Kyoto Encyclopedia of Genes and Genomes (KEGG) database at https://www.genome.jp/kegg/ (accessed on 1 November 2021).Isolate W30 was purified through the plate streaking method on LB solid medium at 30 °C. Cells were fixed with 2.5% glutaraldehyde (pH 6.8) at 4 °C overnight and dehydrated for 20 min each in an ascending ethanol dehydration series . Cells were dried through a CO7F/1492R  based onnapA, narZ, narH, nirK, nirS, norB and nosZ which were supposed in the genome of W30 were amplified with corresponding primer pairs .HN-AD related genes including er pairs . The PCR600 value of each mixture was determined by a spectrometer . The initial OD600 values were 0.148 ± 0.005 for HNM, 0.151 ± 0.007 for DM1, and 0.153 ± 0.004 for DM2. The flasks were incubated at 30 °C for 24 h with shaking at 150 rpm to provide 6.1 mg/L dissolved oxygen in the system. OD600, NH4+-N, NO3−-N, NO2−-N, dissolved oxygen (DO), temperature, and Chemical Oxygen Demand (COD) were assayed every 3 h. Total nitrogen (TN) and dissolved total nitrogen (DTN) were tested at 0 h and 24 h, respectively. Except for OD600 and TN, the rest of the parameters were measured by filtering each mixture with a 0.45 μm filter . NH4+-N was measured by the colorimetric method with Nessler’s reagent at 420 nm. NO3−-N was determined by the phenoldisulfonic acid method. NO2−-N was analyzed by the colorimetric method with N--diaminoethane. TN and DTN were estimated using the alkaline potassium persulfate digestion-ultraviolet spectrophotometric method . COD was determined using the dichromate method. Methods for chemical quantification were referred to standard methods [A heterotrophic nitrification medium (HNM) and two aerobic denitrification media (DM1 and DM2) were prepared as basic media for characterizing W30′s N removal capacity under oxic conditions ,29,30. A methods  if not o3−-N removal capacity under oxic condition. The required C/N ratios were achieved by changing the content of sodium citrate in the system. The initial NO3−-N concentration was set as 50 mg/L. The initial pH was 7.0. The initial inoculation volume was 1% (v/v). The cell culture was incubated at 30 °C for 24 h with shaking (150 rpm). Samples were taken and tested at 0 h and 24 h.A series of C/N ratios (1–20) based on DM1 were investigated to illustrate their effects on W30′s NO3−-N removal capacity under oxic condition. The initial NO3−-N concentration was set as 50 mg/L. The C/N ratio was adjusted to 8. The initial pH was 7.0. The initial inoculation volume was 1% (v/v). The cell culture was incubated at 30 °C for 24 h with shaking (150 rpm). Samples were taken and tested at 0 h and 24 h.Five carbon sources including sucrose, sodium citrate, glucose, sodium acetate, and sodium bicarbonate based on DM1 were investigated to illustrate the effects of different carbon sources on W30′s NO3−-N removal capacity under different DO concentrations. The different levels of dissolved oxygen in the media were achieved using various rotation speeds (from 30 rpm to 180 rpm) of a thermostat vibrating incubator  [3−-N concentration was set as 50 mg/L. The C/N ratio was adjusted to 8. The initial pH was 7.0. The initial inoculation volume was 1% (v/v). The cell culture was incubated at 30 °C for 24 h. Samples were taken and tested at 0 h and 24 h.Six levels of dissolved oxygen  based on DM1 were investigated to illustrate their effects on W30′s NO, China) ,32. The 3−-N removal capacity under oxic condition. The required pH values were achieved by adjusting the buffering ratio of 0.5 mol/L NaOH and 0.5 mol/L HCl in the system. The initial NO3−-N concentration was set as 50 mg/L. The C/N ratio was adjusted to 8. The initial inoculation volume was 1% (v/v). The cell culture was incubated at 30 °C for 24 h with shaking (150 rpm). Samples were taken and tested at 0 h and 24 h.A series of initial pH values (3–12) based on DM1 were investigated to illustrate their effects on W30′s NO3−-N removal capacity under oxic condition. The temperature range (15–45 °C) with a 5 °C ascending was achieved by setting the incubation temperature of the thermostat vibrating incubator . The initial NO3−-N concentration was set as 50 mg/L. The C/N ratio was adjusted to 8. The initial pH was 7.0. The initial inoculation volume was 1% (v/v). The cell culture was incubated for 24 h with shaking (150 rpm). Samples were taken and tested at 0 h and 24 h.A series of temperatures (15–45 °C) based on DM1 were investigated to illustrate their effects on W30′s NO3−-N removal capacity under oxic conditions. The different inoculation volumes were achieved by adjusting the amount of the cell culture amendment. The series of inoculation volume was set as 0.1%, 0.2%, 0.5%, 1.0%, and 1.5% (v/v). The initial NO3−-N concentration was set as 50 mg/L. The C/N ratio was adjusted to 8. The initial pH was 7.0. The cell culture was incubated at 30 °C for 24 h with shaking (150 rpm). Samples were taken and tested at 0 h and 24 h.A series of inoculation volumes based on DM1 were investigated to illustrate their effects on W30′s NO3. Continuous aeration (two hours) was followed by an hour of rest. The NO3−-N concentration in the effluent of the MBR was 58.65 ± 1.34 mg/L. Four treatments were conducted with the effluent to explore W30′s N removal capacity under oxic condition (6.1 mg/L DO) including (i) blank control (no W30 and carbon amendment); (ii) 1% (v/v) W30 suspension inoculation; (iii) 1% (v/v) W30 suspension inoculation and sodium citrate amendment, up to 400 mg/L carbon in the system; (iv) 1% (v/v) W30 suspension inoculation and sodium citrate amendment, up to 800 mg/L carbon in the system. Each treatment had three replicates. The incubations were carried out at 30 °C for 24 h with shaking (150 rpm). Samples were taken and tested at 0 h and 24 h.An assembled MBR with a biological treatment unit and a membrane unit for separation (pore size: 10 nm) was applied for the effluent assay. The aim of this study was to evaluate the feasibility of using isolate W30 for removing high N content wastewater after MBR treatment when the primary N removal efficiency was low. The influent was domestic wastewater. The daily wastewater treatment capacity of this MBR was about 1.62 mThe removal efficiency (RE) and the removal rate (RR) of N compounds under different conditions were calculated according to the following formula :Removal 0 h and Ct h were the initial and final concentrations of each N compound such as NH4+-N, NO3−-N, and NO2−-N at 0 h and t h, respectively.In which, CThe cellular N content (biomass produced) was calculated by subtracting cellular-N at 0 h from cellular-N at 24 h ,33:CelluGaseous N content (N loss) was calculated by subtracting TN at 24 h from TN at 0 h as the following formula, or, the produced Gas-N can be also calculated from the parameter DTN:The denitrification and assimilation efficiency of W30 on N transformation was calculated by the following formula:TN removal efficiency was estimated by summing denitrification and assimilation efficiency as the following formula:One-way analysis of variance (ANOVA) with Tukey method was used to evaluate the statistical significance of environmental constraints on W30′s N removal capacity under oxic condition. Data are shown as mean ± standard deviation.Pannonibacter sp. isolate W30 was deposited in GenBank under accession number KT380575.1. The sequenced functional gene series nosZ, nirK, norB, narZ, and narH were deposited in GenBank under accession numbers from OK431598 to OK431602.The sequenced partial 16S rRNA gene of −1 dilution of the cultured Liangshui River sample. One of the aerobic denitrifiers, isolate W30, could form a yellowish, circular, and convex colony with a smooth surface and entire edge on a LB plate. The colony was opaque and sticky  (Alphaproteobacteria. The phylogenetic tree based on evolutionary distance (neighbor-joining) showed that the HN-AD microorganisms were quite diverse, distributed among at least 13 different families. The genus Pannonibacter comprised several species such as P. phragmitetus, P. indicus, and P. carbonis. Although isolate W30 was grouped within Pannonibacter and close to Pannonibacter phragmitetus, it detached slightly from the internal clusters of the genus. As observed in the phylogenetic tree HI, napAB, nirK, nirS, norBC, and nosZ. An entire series of reduction genes targeting substances from nitrate to N2O were detected in Stappiaceae , Burkholderiaceae , and Pseudomonadaceae , which belong to Proteobacteria only. Other HN-AD microbes showed no full capacity to reduce all the intermediates in denitrification.The sequenced partial 16S rRNA gene of W30 1353 bp) was nearly identical (99.78%) to that of the type strain 28009.1) , which btic tree , outsideens LAD9 , Alcalig3 bp was ae CF-S9 , Acinetotrain YB ,37, Pseu YZN-001 , and Bactrain L7 . VariousnapA and nitrite reductase nirS were negatively supported by further sequencing. The results were in line with the N metabolism pathway of Pannonibacter phragmitetus 31801 (GenBank: CP013068) shown in KEGG (3−-N → NO2−-N → NO → N2O → N2) of Pannonibacter sp. W30 under oxic conditions was proposed (The entire series of denitrification genes were successfully amplified from W30′s genome A. Howeve in KEGG B. Therefproposed C.4+-N in HNM with an average rate of 5.18 mg/L/h in 9 h (Ochrobactrum anthropic LJ81 (3.85 mg/L/h) [Vibrio diabolicus SF16 (2.29 mg/L/h) [4+-N was 97.11% with a linear average rate of 10.21 mg/L/h during 3-6 h. At the same time, a NO2−-N anomaly around 0.105 ± 0.005 mg/L (3 h) and 0.127 ± 0.058 mg/L (6 h) appeared but rapidly decreased therefrom. In addition, no obvious amount of NO3−-N appeared during isolate W30-initiated heterotrophic nitrification.Isolate W30 could heterotrophically remove NHh in 9 h D. The re mg/L/h)  and 2.26 mg/L/h) . The rem3 and NaNO2 as the sole N source, respectively. The concentration of NO3−-N was drastically depleted under the oxic condition from 53.43 ± 0.73 mg/L to a concentration below the detection limit in 12 h (Klebsiella pneumonia CF-S9 (8.64 mg/L/h) [2−-N appeared at 6 h and reached 1.913 ± 0.448 mg/L at 12 h, which was in parallel to the linear depletion of NO3−-N. Aerobic utilization of NO2−-N by isolate W30 was also observed (2−-N remained in the system at 24 h (≤0.016 mg/L). The removal efficiency of NO2−-N by isolate W30 in 24 h was 99.98%.Isolate W30′s ability of aerobic denitrification was evaluated using DM1 and DM2 with KNO in 12 h E. The liobserved F. The li4)2SO4 was the sole N source in HNM, the highest TN removal efficiency could reach 91.34%. Relative to NO3−-N (8.74%) and NO2−-N (10.31%), more gaseous N was prone to occur when NH4+-N (26.64%) was the N source. Assimilation was the dominant driver for N depletion in all the systems. There is no marked difference in the denitrification of KNO3 and NaNO2 in DM1 and DM2.Through HN-AD, isolate W30 achieved high TN removal efficiency with various N sources . When  was removed. As the C/N ratio increased to 14, W30 achieved the greatest NO3−-N removal (98.62 ± 1.40%). Increasing the C/N ratio from 14 did not enhance NO3−-N removal efficiency while cell density continually increased.The investigation on the effect of C/N ratio showed that the higher the ratio, the greater the N removal efficiency, until the ratio reached 14 A. The is3−-N removal efficiencies for isolate W30  with no statistical significance. Following that, the NO3−-N removal efficiency decreased as dissolved oxygen concentrations increased from hypoxic condition to oxic condition (5.9 mg/L DO) significantly. Finally, a medium NO3−-N removal efficiency occurred at oxic conditions (DO ≥ 5.9 mg/L).Six dissolved oxygen concentrations were achieved by adjusting the rotation speed from 30 to 180 rpm representing 1.8, 3.1, 4.2, 5.9, 6.1, and 6.2 mg/L DO C. The hi3−-N at low pH  and 15 °C (8.45 ± 6.31%).A linear relationship between temperatures and NOto 30 °C E. The hi3−-N removal efficiencies were proportional to the initial inoculation volumes from 0.1% to 0.5%, whereas no statistical significance was observed from 0.5% to 1.5% (NO to 1.5% F.3−-N (58.65 ± 1.34 mg/L) with a trace amount of NH4+-N and NO2−-N. Isolate W30 showed a high capacity for NO3−-N removal in the effluent under oxic condition if a sufficient carbon source was supplied  and the nitrous oxide reductase gene nosZ (447 bp) were successfully amplified, indicating that W30 could use nitrite as an intermediate electron acceptor for aerobic denitrification [2 during the process. It is noteworthy that among a few known HN-AD microbes belonging to Alphaproteobacteria including Paracoccus, Pannonibacter, and Agrobacterium, Pannonibacter shows an advantage to develop a felicitous technology for the treatment of N-laden wastewater at the full-scale level. According to the N metabolism pathway in KEGG, the type strain Agrobacterium sp. RAC06 has no nosZ in its genome, which may lead to the accumulation of nitrous oxide, a major scavenger of stratospheric ozone. In addition, nirS rather than nirK is in the genome of Paracoccus denitrificans [nirS encodes a homodimer cytochrome cd1-containing nitrite reductase while nirK encodes a copper-containing nitrite reductase [nirS may be sensitive to environmental pH, which limits its application in wastewater treatments. Indeed, it was reported that a greater loss in nirS abundance occurred when the environmental pH was below 4.7 [The development of new wastewater treatment technologies has been influenced by the discovery that HN-AD microorganisms can simultaneously nitrify NH removal . There itetus B1 . Since Oductases , W30′s afication  and nitreductase ,42. The elow 4.7 .Acinetobacter sp. ND7 produced 4.7 mg/L of nitrate from ammonia at the initial 8 h of heterotrophic nitrification, but it delayed till 24 h to consume it completely [3 and NaNO2 were the initial N sources, isolate W30 was apt to store them as cellular nitrogen.Isolate W30 could remove ammonia with a high rate and efficiency 97.11%) by two processes, assimilation into cellular nitrogen and aerobic denitrification (AD). Furthermore, nitrite was found to be a nitrification intermediate during this latter process as it was produced in trace amounts at the initial 6 h and then quickly consumed. This indicated that isolate W30 could transform ammonia to nitrite by heterotrophic nitrification (HN) and then use it for aerobic denitrification (AD). In parallel, no obvious amount of nitrate was detected during isolate W30-mediated HN. However, it was reported that a known HN-AD bacterium % by two Alphaproteobacteria being oligotrophs, isolate W30 seemed to benefit from high C/N ratios during aerobic NO3−-N removal. This result is in line with that of other aerobic denitrifiers such as Citrobacter diversus [Cupriavidus sp. S1 [Alphaproteobacteria, could adapt to a broad and flexible range of C/N ratios. Moreover, W30 was verified a strict heterotroph through the NaHCO3 test. Meanwhile, sucrose was verified not a favorite carbon source for W30. W30′s aerobic N removal efficiency decreased as dissolved oxygen concentration increased. Similarly, an aerobic denitrification bacterium Citrobacter diversus showed the highest denitrification rate when dissolved oxygen was low [3−-N removal efficiency could still be achieved even at oxic conditions, indicating W30 is an aerobic bacterium that could tolerate high O2 concentrations. In addition, W30 exhibited a series of high NO3−-N removal efficiencies from neutral to alkaline pHs. The result is consistent with that of known HN-AD microbes such as Pannonibacter phragmitetus F1 [Pannonibacter isolated from a Hungarian soda lake [3−-N removal efficiencies up to 75.73 ± 6.23% were achieved by W30 at temperatures from 30 °C to 40 °C. As a comparison, the NO3−-N removal efficiency of Acinetobacter sp. ND7 reached only 6.69 ± 0.66% at 40 °C [600 = 0.305) was sufficient for initiating the NO3−-N removal process. Furthermore, Pannonibacter sp. W30 achieved nearly full NO3−-N removal in effluent from the pilot MBR. The C/N ratio, however, seemed critical for isolate W30 to tackle high N content wastewater at the full-scale level.Despite most diversus  and Cuprs sp. S1 . In this was low . For isotetus F1  and a stoda lake . It is boda lake . Moreoveat 40 °C . Our resAlphaproteobacteria Pannonibacter sp. W30 was isolated, identified, and characterized in this study. A potential HN-AD pathway of W30 was proposed. The effect of environmental constraints on aerobic NO3−-N removal was investigated. Based on the results, Pannonibacter W30 was proved to be able to perform N removal from water environments with high inorganic N removal efficiency. The study verified that Pannonibacter sp. W30 contains nirK, norB, and nosZ genes which encode a nitrite reductase, a nitric oxide reductase, and a nitrous oxide reductase. These enzymes can transform NO2−-N to NO, N2O, and N2, avoiding NO2−-N accumulation in the process. Pannonibacter sp. W30 also has a copper-containing nitrite reductase nirK, a relatively robust enzyme under low pH. Our results suggest that although Pannonibacter sp. W30 could be able to conduct aerobic denitrification, dissolved oxygen is still a limiting factor affecting this process. As an aerobic heterotrophic nitrogen removal bacterium, Pannonibacter sp. W30 adapts to a broad range of C/N ratios in wastewaters, which may be feasible for developing novel HN-AD technologies.An aerobic heterotrophic nitrogen removal