Doxorubicin (DOX), which is widely used in cancer treatment, can induce cardiomyopathy. One of the main mechanisms whereby DOX induces cardiotoxicity involves pyroptosis through the NLR family pyrin domain containing 3 (NLRP3) inflammasome and gasdermin D (GSDMD). Increased NAPDH oxidase (NOX) and oxidative stress trigger pyroptosis. Exogenous 8-hydroxydeoxyguanosine (8-OHdG) decreases reactive oxygen species (ROS) production by inactivating NOX. Here, we examined whether 8-OHdG treatment can attenuate DOX-induced pyroptosis in H9c2 cardiomyocytes. Exposure to DOX increased the peroxidative glutathione redox status and NOX1/2/4, toll-like receptor (TLR)2/4, and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) expression, while an additional 8-OHdG treatment attenuated these effects. Furthermore, DOX induced higher expression of NLRP3 inflammasome components, including NLRP3, apoptosis-associated speck-like protein containing a c-terminal caspase recruitment domain (ASC), and pro-caspase-1. Moreover, it increased caspase-1 activity, a marker of pyroptosis, and interleukin (IL)-1β expression. All these effects were attenuated by 8-OHdG treatment. In addition, the expression of the cardiotoxicity markers, atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) was increased by DOX, whereas the increase of ANP and BNP induced by DOX treatment was reversed by 8-OHdG. In conclusion, exogenous 8-OHdG attenuated DOX-induced pyroptosis by decreasing the expression of NOX1/2/3, TLR2/4, and NF-κB. Thus, 8-OHdG may attenuate DOX-induced cardiotoxicity through the inhibition of pyroptosis.The online version contains supplementary material available at 10.1186/s12860-022-00454-1. Despite its effectiveness, DOX can induce serious complications such as cumulative cardiotoxicity , 2. It h2 . However2 , 5. DOX-2 . The pat2 –10. More2 .Damage-associated molecular patterns (DAMPs) are released from dying cells and are recognized by NOD-like receptors such as NLRP3 . The NLRTumor necrosis factor (TNF)-α expression is increased in DOX-induced cardiotoxicity , 27, andEndogenous 8-OHdG is frequently used as an indicator of the oxidative DNA damage induced by ROS, as it is an oxidized nucleoside of DNA . ParadoxHere, we examined whether exogenous 8-OHdG can attenuate DOX-induced pyroptosis in H9c2 cardiomyocytes. We hypothesized that treatment with exogenous 8-OHdG would lead to the downregulation of NOX, and NF-κB expression, and decreased TNF-α production by decreasing Rac1, eventually leading to the attenuation of DOX-induced pyroptosis.H9c2 cells were obtained from ATCC  and grown in Dulbecco’s modified Eagle’s medium (DMEM)  with 10% fetal bovine serum  and 1% penicillin–streptomycin (Welgene). 8-Hydroxydeoxyguanosine (8-OHdG), was provided by Professor Myung-Hee Chung. DOX was obtained from Seleckchem .4 cells were plated in 96-well culture plates, treated with DOX or 8-OHdG, and incubated for 24, 48, or 72 h at 37 °C. Then, the cells were incubated in medium containing 0.5 mg/mL 3--2,5-diphenyltetrazolium bromide (MTT)  for 1 h at 37 °C. Dimethyl sulfoxide (DMSO) was added at the end of the reaction to dissolve the formazan crystals from viable cells. The absorbance was measured at 560 nm with a microplate reader .1 × 104 cells were seeded in 96-well plates and treated with DOX and 8-OHdG in culture medium containing 1% FBS for 24–48 h at 37 °C. Cell-free culture supernatants were mixed with lactate dehydrogenase (LDH) reaction mixture  and incubated for 30 m at room temperature. The absorbance was measured at 490 nm.2 × 10The Rac1 activity was measured using Rac1 activation assay kit  according to the manufacturer’s protocol. Briefly, for immunoprecipitation, 200 μg proteins were incubated in assay buffer containing p21-activated protein kinase (PAK) p21-binding domain (PBD) agarose beads at 4℃ for overnight. After washing with 1X assay buffer, pull-down supernatants were separated in 12% SDS–PAGE gels. Primary antibodies to Rac1 (1:1000) (Cell Biolabs) and β-actin (1:3000) (Santa Cruz Biotechnology) were used. Images were taken with an Amersham Imager 600 .5 cells/60 mm dish) using RNAiso plus  and a Qiagen RNeasy Mini Kit . RNA quantitation was performed using NanoDrop-2000 . Then, cDNA synthesis was performed using a PrimeScript First Strand cDNA Synthesis Kit . Real-time PCR was performed on a CFX96 real-time system  using SYBR Green I Universal PCR Master Mix (Takara Bio) and primers  in reactions with the following conditions: 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s, and 60 °C for 1 min. GAPDH was used as a reference gene. Relative gene expression presents the data of the gene of interest relative to internal control gene using the comparative CT method also referred to as the 2CT−△△ method. Primer  sequences for rat TNF-α (GeneBank Accession no. AB553578.1), NLRP3 (GeneBank Accession no. XM_039085397.1), atrial natriuretic peptide (ANP) (GeneBank Accession no. M27498.1), brain natriuretic peptide (BNP) (GeneBank Accession no. XM_032887581.1), GATA binding protein 4 (GATA4) (GeneBank Accession no. XM_032917655.1), GATA binding protein 6 (GATA6) (GeneBank Accession no. NM_019185.2), TLR-2 (GeneBank Accession no. NM_198769.2), TLR-4 (GeneBank Accession no. NM_019178.2), and GAPDH (GeneBank Accession no. NM_017008) are listed in Table Total RNA was isolated from H9c2 cells (3 × 105 cells/60 mm dish) were lysed in RIPA buffer (20 mM Tris–HCl (pH 7.5), 150 mM NaCl, 1 mM EGTA, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM sodium orthovanadate, 1 μg/ml leupeptin, 1% sodium deoxycholate, 1% NP-40) . 10–20 μg proteins were separated in 8–12% SDS–PAGE gels and transferred onto PVDF membranes. After blocking with 5% skim milk, the membranes were incubated with primary antibodies at 4℃ for overnight. Primary antibodies for ASC (1:1000) (GeneBank Accession no. NM_172322.1) , caspase-1 (1:1000) (GeneBank Accession no. NM_012762.3) , caspase 3 (1:1000) , IL-1β (1:1000) (GeneBank Accession no. XM_032902343.1) , NOX1 (1:1000) (GeneBank Accession no. NM_053683.2) , NOX2 (1:1000) (GeneBank Accession no. NM_023965.1) , NOX4 (1:1000) (GeneBank Accession no. NM_053524.1) , NF-κB p65 (1:2000) (GeneBank Accession no. AJ002424.2) , Phospho-NF-κB p65 (1:1000) , GSDMD-NT (1:1000) , and β-actin (1:3000)  were used. Images were taken with an Amersham Imager 600 . Band intensities were normalized to β-actin using ImageJ software .Cells  as per the manufacturer’s instructions. First, cells were incubated in medium containing fluorochrome-labeled inhibitors of caspases (FLICA) for 4 h at 37 °C in the dark. Then, the cells were washed and transferred to black microtiter plates to measure fluorescence intensity  with a microplate reader (Molecular Devices). Then, cells were fixed with fixation buffer for 2 min, washed with Dulbecco’s phosphate-buffered saline (DPBS), placed on a glass slide to which 50 μL antifade mounting medium  was applied, and covered with 25 mm cover glasses. Images were taken with an LSM 700 Zeiss confocal microscope  and analyzed using ZEN software (Carl Zeiss). The experiment was performed three times.3 × 106 cells using lysis buffer was incubated in medium containing thiol or GSSG probe at room temperature for 10–60 m. Then, we measured concentrations of GSH and total glutathione (GSH + GSSG) at Ex/Em = 490/520 nm using a fluorescence microplate reader . The GSH/GSSG ratio was calculated from concentrations of GSH and GSSG.Cells were plated in 100 mm dishes and treated with DOX and 8-OHdG for 24 h. A glutathione assay kit  was used as per the manufacturer’s instructions. Briefly, cell suspension from lysed 10P < 0.05, **P < 0.01, and *** P < 0.001 in all experiments.All experiments were repeated more than three times, and the average values are presented unless otherwise stated. Data are presented as mean ± standard deviation. The statistical significance of the results was determined with Prism® software  using one-way ANOVA and post-hoc Dunnett’s test. Significance was defined as *First, we performed preliminary experiments to determine the appropriate toxic concentration of DOX and the effective concentration of 8-OHdG. Cells were treated with 0.1, 1, 5, 10, and 20 μM DOX or 25, 50, 100, 250, 500, and 1000 μg/mL 8-OHdG for 24, 48, and 72 h. Then, cell viability was evaluated with the MTT assay.50 values of DOX over the study period were 20.6 μM (24 h), 0.4778 μM (48 h), and 0.02895 μM (72 h). After 24 h culture, the viability of 8-OHdG-treated H2c9 cells did not significantly differ from that of the control when the 8-OHdG concentration ranged from 0 to 250 μg/mL; however, cell viability was significantly decreased when the concentration was ≥ 500 μg/mL , 595.5 μg/mL (48 h), and 544.4 μg/mL (72 h). To evaluate the effect on pyroptosis, we used 1 μM DOX as in previous reports [The results showed that cell viability gradually decreased as the concentration of DOX increased Fig. A. The IC/mL Fig. B. After /mL Fig. B. The IC reports . In addiCytotoxicity of DOX-treated H9c2 cells was reduced by 8-OHdG treatment Fig. . Thus, wGATA4 and GATA6 are transcription factors involved in cell survival. We assessed the effect of DOX and 8-OHdG on the expression of GATA4 and 6 in cardiomyocytes. As shown in Fig. Given that 8-OHdG is involved in oxidative stress via Rac1, we examined the regulatory effects of DOX and 8-OHdG on expression of NOX1/2/4 and the Rac1 activity in H9c2 cells.We performed the Rac1 activation assay using immunoprecipitation and found that 250 μg/mL 8-OHdG decreased the rat Rac1 activity in DOX-exposed H9c2 cells Fig. A. AdditiOxidative stress is the result of both the increased production of ROS and decreased content of endogenous antioxidants such as CAT, SOD, and GSH in the cellular system , 33. ThuDOX-induced cardiotoxicity increases TNF-α, leading to pyroptosis –28. ThusTo evaluate whether 8-OHdG decreased NOX1/2/4 and TLR2/4 similar to Rac1 siRNA, H9c2 were transfected with siRNAs for negative control and two kinds of rat Rac1, and then treated with DOX and/or 8-OHdG for 24 h. The inhibitory effects of 8-OHdG on the regulation of NOX1/2/4, and TLR2/4 in DOX-exposed H9c2 cells were similar to that of two kinds of Rac1 siRNA  by DOX was reduced by 54.8–91.2% in additional treatment of 8-OHdG  of more than 10% to 50% . DOX-indThe prognosis of DOX-induced congestive heart failure is very poor . To prevThe NLRP3 inflammasome has been shown to be involved in DOX-induced cardiotoxicity. DOX treatment increases NLRP3 inflammasome and IL-1β secretion in the myocardium of mice . IncreasIncreased ROS or NOX activity is known to increase NLRP3 inflammasome , 35. ExoIn our study, 8-OHdG decreased Rac1 activity in DOX-treated H9c2 cells  and caspase-1, whereas 8-OHdG treatment decreased it. DOX treatment led to increased expression of both pro- IL-1β and IL-1β in H9c2 cells. GSDMD-NT levels were increased by DOX treatment and decreased by 8-OHdG treatment. Moreover, the amount of pyroptotic cells as evaluated by a caspase-1 activity assay was increased by DOX treatment and decreased by 8-OHdG treatment.Both ANP and BNP are known to be useful predictors and prognostic markers of heart failure –62. ANP In our study, the expression of ANP and BNP was increased by DOX treatment and decreased by 8-OHdG treatment. However, the expression of GATA4/6 was decreased by DOX treatment, and 8-OHdG treatment only increased GATA6 expression. Thus, it seems that 8-OHdG decreases the expression of cardiotoxicity makers in DOX-treated H9c2 cells which was accompanied with decreased expression of TLR2/4, NF-κB, and NOX 1/2/4 and pyroptosis. DOX-treated H9c2 cells also exhibited increased expression of IL-6 which induced cell injuries as well as IL-1β or TNF-α . Since eImmune checkpoint inhibitors (ICIs) which show effect of anti-PD-1 (nivolumab and pembrolizumab), anti-PD-L1 , and anti-CTLA-4 antibodies (ipilimumab and tremelimumab) –75 also Collectively, exogenous 8-OHdG decreased the expression of NOX1/2/4, TLR2/4, and NF-κB by decreasing Rac1 activity in DOX-treated cells, which eventually led to reduced NLRP3 inflammasome, IL-1β, and GSDMD-NT levels. Furthermore, the decrease in the levels of pyroptosis-related factors led to the decreased expression of myocarditis-related markers such as ANP and BNP Fig. D. Thus, Additional file 1.


Meretrix lusoria and used in this investigation shown strong antithrombotic action. UV–Vis, FT-IR, SAX-HPLC, and NMR were used to explore the structural characteristics of M2, results indicated that M2 similar with heparin, its average molecular weight was 22.58 kDa. Which was primarily made up of→4)-α-IdoA2S-(1→4)-α-GlcNS6S-(1→ (31.19%), →4)-β-GlcA-(1→4)-α-GlcNAc (1→ (23.21%), →4)-β-GlcA-(1→4)-α-GlcNS (1→ (13.87%), →4)-α-IdoA2S-(1→4)-α-GlcNS (1→ (8.95%), →4)-β-GlcA-(1→4)-α-GlcNAc6S (1→ (7.39%) and →4)-β-GlcA-(1→4)-α-GlcNS6S (1→ (7.63%). The antithrombotic activity of M2 was evaluated using measurements of the anticoagulant effect in vitro and the fibrinolytic capability in vitro and in vivo, and M2 has 122.4 U/mg of anticoagulant activity and 1.41 U/mg of fibrinolytic activity, respectively. Additionally, a mouse tail-cutting model was used to assess the bleeding effect in real time, it found that M2 had a reduced hemorrhagic risk than heparin. Consequently, M2 could be exploited to develop functional foods or medications with antithrombotic properties.Heparinoid, a sulfate polysaccharide derived from marine organisms was attracted largely attention due to its versatile activities. A naturally occurring heparinoid (M2) that was extracted from the mollusk   •A natural heparinoid (M2) was discovered in the seafood Meretrix lusoria.•M2 has slightly lower anticoagulant activity than mammalian heparin but a weaker bleeding effect.•M2 was found to have an active effect on the fibrinolysis pathway in vitro and in vivo.•M2 considered replacing mammalian heparin with functional foods or drugs with anti-thrombotic effects. Rabbit plasma, bovine plasma fibrinogen, urokinase (50000 U/g), thrombin (300 U/mg), alkaline protease 2709 and papain were gifts from Shanghai yuanye Bio-Technology Co. Ltd . Thrombin time (TT), prothrombin time (PT) and activated partial thromboplastin time (APTT) test kits were obtained from Sun Bio-Technology Co. Ltd. . Urokinase-type plasminogen activator (u-PA), tissue-type plasminogen activator (t-PA), and plasminogen activator inhibitor (PAI-1) were obtained from Shanghai Lengton Bioscience Co., Ltd . Standard monosaccharide references were prepared from Sigma-Aldrich (Germany). Heparin standards  were obtained from Adhoc Bio-Technology Co., Ltd . Heparinlyase I, II, III were purchased from Asnail Biotechnology Co. Ltd. . Unless specified otherwise, all chemicals were of analytical grade or HPLC grade.2.2Sprague-Dawley (SD) female rats, weighing 220–250 g, were acclimated to the laboratory for at least 7 days prior to surgery or experimentation. All experiments were carried out according to the NIH. .2.3M. lusoria was obtained from Zhanjiang Dongfeng Market, China. Dried tissue was obtained after homogenizing the soft tissue and freeze-drying. 100 g of this dry tissue were mixed with 10 times volume distilled water in high-speed tissue masher, and pH adjusted to 8.0. Then, NaCl (6 mg/mL), Papain (5.4 mg/mL) and 2709 alkaline protease (5.4 mg/mL) were put in and hydrolyzed for 20 h (50 °C). Next, the hydrolysate was boiled for 10 min, centrifuged  after cooling, and the supernatant was obtained. The supernatant was transferred to a column (26 cm × 100 cm) filled with AMBERLITE FPA98 Cl macro porous ion exchange resin. Dynamic adsorption was carried out by constant flow pump at 45 °C, and the adsorption rate was 10 mL/min. After adsorption, gradient elution was performed with 0, 1.0, 1.5, and 3.5 mol/L NaCl solution in sequence. Determination of glycosaminoglycan content in each fraction by Alcian blue method. Fractions eluted with 1.0 mol/L, 1.5 mol/L, and 3.5 mol/L NaCl were collected and precipitated by 0.4 vol of ethanol overnight. The precipitate was collected by centrifugation, dissolved in distilled water, dialyzed for 48 h at 4 °C, and lyophilized. The elution components of 1.0 mol/L, 1.5 mol/L, and 3.5 mol/L NaCl were named M1, M2, and M3, respectively.The clam 2.4Heparin anticoagulant potency was evaluated by comparing with the concentration necessary to prevent the clotting of rabbit plasma using CHP (Chinese Pharmacopoeia) method, and heparin standard (197 U/mg) was used as control; Heparin content was determined by methylene blue spectrophotometry with heparin as standard; Protein content was measured by Folin-phenol method with bovine serum protein as standard; Uronic acid was assayed by the carbazole-sulfuric acid method with glucuronic acid as standard; Glucosamine content was determined by Elson-Morgan method with glucosamine sulfate as standard; Sulfate groups was determined by barium sulfate turbidimetric using potassium sulfate as standard, as previously described .2.5The sample and standard are dissolved by distilled water to prepare 3 mg/mL. The acetate film (8 × 12 cm) was immersed in electrophoresis buffer for 30 min, and the surplus buffer was lightly absorbed by filter paper. Samples were added to the matte surface of the acetate film and electrophoresed at 7 mA for 30 min. Then, the film was dyed in 0.5% Alcian blue-acetic acid for 25 min, and then decolorized with 2% acetic acid for 25 min .2.62.6.1−1 wavenumber range using a Bruker Tensor 27 FTIR spectrometer.Samples were dried and then mixed with KBr followed by compression. Then, Fourier transforms infrared (FT-IR) spectra were conducted within the 4000–500 cm2.6.2The purity and molecular weight (Mw) of the samples were determined by high-performance gel permeation chromatography (HPGPC) on a Waters Ultra hydrogel column 500  and a refractive index detector (Agilent 1200). The Mw of samples was calculated from a standard curve based on reference heparin  .2.6.3The M2 monosaccharide composition was detected by 1-phenyl-3-methyl-5-pyrazolone and high performance liquid chromatography (PMP-HPLC). M2 was hydrolyzed with trifluoroacetic acid  at 110 °C for 6 h and then derivatization was conducted PMP. After reaction ending, the excess PMP was extracted with 2 mL chloroform, and the supernatant was filtered. Then sample was analyzed by Agilen1200 liquid chromatography according to the retention time of monosaccharide standards.2.6.4Disaccharide composition of M2 was assessed by strong anion exchange-high-performance liquid chromatography (SAX-HPLC) on waters spherisorb SAX column  at 234 nm wavelength, after the sample was enzymatically digested with heparin lyase I, II, III . Besides2.6.51H-NMR,13C-NMR, 1H-1H COSY, 1H-1H TOCSY, and 1H-1H NOESY spectroscopy analyses by Bruker Ascend 700M spectrometer at 25 °C.The M2 sample (50 mg) was soluble in 1 mL of Deuterium oxide  and performed the 2.7M2 hemorrhagic effect was induced by transection of the tail in rats . Rats we2.8The samples were formulated with normal saline to prepare solutions of different concentrations . Measurements of PT, APTT and TT were performed according to the manufacturer's recommended protocols .2.92.9.1in vitro was tested using the fibrin plate method (in vitro of the sample (12 mg/mL) was computed according to the standard curve and the corresponding dissolving area of the sample.Fibrinolysis e method . Briefly2.9.2SD rats were acclimated to the laboratory for at least 7 days prior to surgery or experimentation. They were optionally split into seven groups of 6 rats each and fasted overnight prior to the experimentation. They were anaesthetized with chloral hydrate and injected with samples , heparin  and saline via the femoral vein. After 2 h of administration, blood was collected by eyeball blood extraction method, coagulated naturally at room temperature for 10–20 min, centrifuged for about 20 min (2000–3000 r), and the upper serum was taken for standby. The levels of serum t-PA, u-PA and PAI-1 in the serum were measured using commercial ELISA kits according to the instructions of the manufacturer .2.10The data were expressed as means ± standard deviation (SD) and analyzed using JMP 10 software. A completely randomized design (CRD) was followed for the designing of the experiments and the data were analyzed by ANOVA .33.1M. lusoria by enzymatic hydrolysis, anion exchange chromatography and alcohol precipitation. Although be obtained from the same clam, there were significant differences in the yield and chemical characteristics of the three fractions . The potency of M2 was about 122.4 U/mg, which was significantly higher than that of M1 (48.0 U/mg) and M3 (85.2 U/mg). The anticoagulant activity of heparin-like compound (M2) from M. lusoria was found to be 122.4 U/mg, which was lower than the purified heparin from pig mucosa (180 U/mg)  were extracted from ractions . The yie80 U/mg)  and high80 U/mg) A. Theref3.2−1 (O-H band) and 2929.01 cm−1 (C-H band)  . Besidesose ring .3.3The Mw of M2 was determined by HPGPC . In Fig.3.4The monosaccharide composition of M2 was detected by PMP-HPLC method D. Glucos3.5M2 unsaturated disaccharides composition was assessed by strong anion exchange-high-performance liquid chromatography (SAX-HPLC) chromatography  Pan et . The cla3.61H-NMR spectral signal of M2 is mainly distributed in three regions. The first is the anomeric carbon region ranged from 4.5 to 5.6 ppm, which contains the signal peaks of N, 6-disulfate glucosamine , 2-disulfate alduronic acid , glucuronic acid  and IdoA2S . Besides, there is a peak with the strongest proton signal at 4.70 ppm, which is the solvent peak produced by deuterated water -α-IdoA2S-(1 →4) -α-GlcNS6S-(1→ (31.19%), →4)-β-GlcA-(1→4)-α-GlcNAc(1→ (23.21%), →4)-β-GlcA-(1 →4)-α-GlcNS(1→ (13.87%), →4)-α-IdoA2S-(1 →4)-α-GlcNS(1→ (8.95%), →4)-β-GlcA-(1 →4)-α-GlcNAc6S (1→(7.39%) and →4)-β-GlcA -(1→4)-α-GlcNS6S(1→ (7.63%).3.7M. lusoria. As shown in p < 0.05). The degree of bleeding in the M2 group was concentration-dependent and lower than that of the HP group (p < 0.05). Furthermore, the highest dose group (80 mg/kg) of M2 still showed a hemorrhagic effect about two times lower than that of the HP group. Therefore, heparinoid from M. lusoria exhibits a weaker bleeding effect when compared to mammalian heparin. Heparin isolated from an ascidian showed comparable antithrombotic activity in an arterial animal model and lower bleeding effects compared to mammalian heparin , which was drawn by the dissolved circle area of different concentrations of urokinase  from hrombin Ⅲ. For anoHeparin in marine species has been found to differ from heparin in mammalian organisms, primarily in terms of molecular weight and sulfation pattern, and to have distinct structures . The dis4M. lusoria heparin analogue is 122.4 U/mg, which plays an anticoagulant role primarily mediated by intrinsic and common coagulation pathway. In addition, the fibrinolytic activity of M. lusoria heparin analogue is 1.41 U/mg, which mainly through promoting the release of t-PA. Although the anticoagulant activity of heparinoid from M. lusoria is lower compared with that of mammalian heparins, the hemorrhagic reaction caused by the analogue is much lower than that of mammalian heparins. The fibrinolytic activity of M. lusoria analogue is slightly higher than that of mammalian heparins. Therefore, heparinoid from M. lusoria exposes mild anticoagulant activity, low bleeding effect and strong fibrinolytic activity. M2 can be considered as an alternative for using mammalian heparin, and developing functional foods or drugs with antithrombotic effects.It was concluded that M2 is a polysaccharide mixture with similar characteristics to heparin. The extraction rate of M2 is 0.53 mg/g (dry weight) and the average molecular weight is 22.58 kDa. Besides, it is majorly composed of →4)-α-IdoA2S-(1 →4)-α-GlcNS6S-(1→ (31.19%), →4)-β-GlcA-(1 →4)-α-GlcNAc (1→ (23.21%), →4)-β-GlcA-(1 →4)-α-GlcNS (1→ (13.87%), →4)-α-IdoA2S-(1 →4)-α-GlcNS (1→ (8.95%), →4)-β-GlcA-(1 →4)-α-GlcNAc6S (1→(7.39%) and →4)-β-GlcA-(1 →4)-α-GlcNS6S(1→ (7.63%). The anticoagulant potency of Jing Chen: Conceptualization, Methodology, Data curation, Formal analysis, Writing – original draft. Zhenxing Du: Conceptualization, Methodology, Data curation, Formal analysis, Writing – original draft. Bingbing Song: Methodology, Writing – review & editing. Rui Li: Methodology, Writing – review & editing. Xuejing Jia: Visualization. Jianping Chen: Methodology, Writing – original draft. Xiaofei Liu: Methodology, Writing – review & editing. Saiyi Zhong: Methodology, Software, Writing – review & editing, Supervision, Project administration, Funding acquisition.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


N′-[(E)-2-hydroxybenzylidene]hydrazinecarboxylate and benzyl N′-[(E)-5-bromo-2-hydroxybenzylidene]hydrazinecarboxylate are presented.The syntheses and low-temperature (90 K) crystal structures of benzyl  N′-[(E)-2-hydroxybenzylidene]hydrazinecarboxylate, C15H14N2O3 (I) and benzyl N′-[(E)-5-bromo-2-hydroxybenzylidene]hydrazinecarboxylate (II), C15H13BrN2O3, have been synthesized by the reaction of either 2-hydroxybenzaldehyde or 5-bromo-2-hydroxybenzaldehyde with benzyl carbazate, respectively. Both the compounds crystallize in the monoclinic crystal system with space groups Pn  and P21/c . Molecular conformations in each structure are similar, and both structures feature strong intramolecular O—H⋯N hydrogen bonds, which form S(6) ring motifs. There are also strong N—H⋯O and weak C—H⋯O hydrogen bonds in both structures, but their modes of packing within their respective crystals are markedly different. Some comparisons are made with the structures of a few related compounds.Benzyl  There are no unusual bond lengths or angles in either structure. The molecules have strong intramolecular O—H⋯N hydrogen bonds (Tables 1S(6) ring motifs , 0.0905 Å (IIA), 0.0692 (IIB)]. These form dihedral angles of 79.92 (3)°, 79.74 (4)°, and 74.27 (4)° to the benzyl groups of I, IIA, and IIB, respectively. Indeed, the V-shaped conformations of IIA, and IIB are strikingly similar, with I only deviating to any appreciable degree at the benzyl group, as evidenced by an overlay of the three molecules  Fig. 1 and benzI) Fig. 2 each con Tables 1 and 2 ▸,3.intramolecular hydrogen bonds in I and II, the structures both feature strong N—H⋯O and weaker C—H⋯O intermolecular hydrogen bonds. These interactions are summarized in Tables 1In addition to the strong O—H⋯N I, the V-shaped  chains  make hydrogen bonds to 21-screw-related copies of themselves via strong (N2—H2N⋯O1) and weak (C3—H3⋯O2 and C6—H6⋯O3) hydrogen bonds  pairs of IIA molecules have close contacts of 3.3379 (9) Å between their Br1A atoms and the centroid of the inversion-related C10A–C15A ring. There is no corresponding close contact for the IIB molecule  group is attached to the hydrazine. QAVFAY features a four-membered 1,2-diazete ring, with the phenyl group fluorinated at its 4-position. Structures GEZTUD and PIVKUD each feature pyrazole rings; the former having a 2,2,2-trifluoroethyl group attached to the pyrazole and a methyl at the 4-position of the phenyl ring, and the latter having a 3,4,5-trimethoxyphenyl attached to its pyrazole ring.A search of the Cambridge Structure Database -benzyl 2-[1-(pyridin-3-yl)ethylidene]hydrazine-1-carboxylate (JENFEQ), (E)-benzyl2-[1-(pyridin-4-yl)ethylidene]hydrazinecarboxylate (JENFIU)  (for I) or 5-bromo-2-hydroxybenzaldehyde  (for II) and benzyl carbazate  were dissolved in methanol (25 ml) and stirred for 3 h at room temperature. The resulting solids were filtered off and recrystallized from ethanol to give I and II with yields of 80% in both cases. The general reaction scheme is summarized in Fig. 10I and II were obtained by slow evaporation of methanolic solutions at room temperature (m.p.: 400–402 K for I and 468–470 K for II).Preparation of 6.I and II were each secured on the tips of fine glass fibres held in copper mounting pins. The crystal of I was mounted from a shallow liquid-nitrogen dewar using tongs first developed for protein cryocrystallography  were collected with the crystals held at 90.0 (2) K. Determination of the absolute structure for I was inconclusive via traditional full-matrix refinement of Flack’s parameter [x = −0.08 (18); Flack & Bernardinelli, 1999y = 0.00 (8); Hooft et al. ; Parsons et al., 2013PLATON  parameters were set to values of either 1.2Ueq  or 1.5Ueq (O—H) of the attached atom.Crystals of  al. 2008, as calc10.1107/S2056989022009100/yk2176sup1.cifCrystal structure: contains datablock(s) I, II, global. DOI: 10.1107/S2056989022009100/yk2176Isup4.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989022009100/yk2176IIsup5.hklStructure factors: contains datablock(s) II. DOI: 2206989, 2206988CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Mycobacterium tuberculosis (Mtb) infection alters the phenotype and function of lung IL-18Rα+ ILC toward a protective interferon-γ-producing ILC1-like population. This differentiation is controlled by type 1 cytokines and is associated with a glycolytic program. Moreover, a BCG-driven type I milieu enhances the early generation of ILC1-like cells during secondary challenge with Mtb. Collectively, our data reveal how tissue-resident ILCs adapt to type 1 inflammation toward a pathogen-tailored immune response.Tissue-resident innate lymphoid cells (ILCs) regulate tissue homeostasis, protect against pathogens at mucosal surfaces, and are key players at the interface of innate and adaptive immunity. How ILCs adapt their phenotype and function to environmental cues within tissues remains to be fully understood. Here, we show that   •+ ILCType 1 inflammation shapes the fate of lung IL-18Rα•+ ILC give rise to protective ILC1-like cells during Mtb infectionIL-18Rα•ILC1-like cells depend on glycolysis for their differentiation and function•BCG vaccination promotes the early generation of ILC1-like cells in the lung Mycobacterium tuberculosis infection, lung ILC precursors differentiate into IFN-γ, producing ILC1-like cells. ILC1-like cells display the protective capacity to control the mycobacterial infection.In this study, Corral et al. demonstrate that the inflammatory and metabolic environments shape the fate of lung ILC precursors expressing IL-18Rα. During  Innate lymphoid cells (ILCs) are a population of tissue-resident cells that play a key part in tissue homeostasis and immunity . ILCs arin situ “ILCpoiesis”  infection induces prolonged proinflammatory responses that are associated with oxidative stress. Mtb infection is associated with metabolic changes in the lungs, involving the utilization of aerobic glycolysis primarily instead of oxidative phosphorylation (OXPHOS) in mitochondria (Warburg effect)  . The inf effect)  in placeMtb infection, we explored how lung ILCs respond and adapt to type 1 inflammation driven by chronic pulmonary infection. Our work uncovers the local differentiation of lung IL-18Rα+ ILCs into a protective ILC1-like population through an IFN-γ/Stat-1-dependent and metabolic reprogramming during Mtb infection.Here, using the murine model of Mtb reference strain H37Rv. At steady state, bona fide lung ILCs were defined as a population that does not express lineage markers  but highly express CD90.2 (high) was identified after the exclusion of ILC1  and ILC3 (RORγt+) cells , like ILC2 precursors from the bone marrow, confirming their immature profile, regardless of ST2 or Arg1 expression  cells A. At ste+) cells , the lun+) cells A and 1B.+) cells B, a phen+) cells . Based oRα− ILC2 A. IL-18Rand Arg1 A. At thedid ILC1 B. In lindid ILC1 , we founpression C. Mtb inand ILC3 D. Of pare cells” A and 1B,  mice with ROSA26fl/stopYFP mice to enable fate mapping of mature ILC2 (+ ILCs and ILC1-like cells expressed IL-5 and yellow fluorescent protein (YFP) after Mtb infection, compared to IL-18Rα− ILC2  in IL-33-treated mice and ILC1-like markers  in IL-12/IL-18-treated animals, the combination of IL-12/IL-18 with IL-33 led to a mixed ILC1/ILC2 phenotype with the capacity to produce both IL-5 and IFN-γ  compared to single IL-5+ or IFN-γ+ producers  method  method , which a , indicating that a lung ILCP skewed toward an ILC2 phenotype. Furthermore, IL-18Rα+ ILCs display a hybrid phenotype between ILC1 and ILC2 in the presence of ILC1-  and ILC2-  activating cytokines. These results sustain the notion that local ILC precursors in the lungs may undergo a context-dependent ILCpoiesis .Further information and request for resources and reagents should be directed to and will be fulfilled by the lead contact, Dan Corral .Rag2−/− (B6.129-Rag2tm1Fwa), STAT1−/− (B6.129S(Cg)-Stat1tm1dlv/J), Red5 mice (B6(C)-Il5tm1.1(icre)Lky/J)n, Rag2−/−γc−/−  on a C57BL/6 J were bred in our animal facility. ROSA26 fl/stopYFP mice (B6.129X1-Gt(ROSA)26Sortm1(EYFP)Cos/J; 006148) were purchased from The Jackson Laboratory through Charles Rivers Laboratory France. IL-12p40−/− mice were obtained through the NIAID-Taconic exchange program. Inducible deletion of mTOR in the ILC lineage was carried out using mice containing Id2creERT2 (JAX stock #016222) (Rosa26tdRFP (fl/fl mice (JAX stock #011009) . #016222)  and Rosaa26tdRFP  alleles a26tdRFP  - in com#011009) . All micMtb, H37Rv, was grown at 37°C in Middlebrook 7H9 medium (Difco) supplemented with 10% albumin-dextrose-catalase  and 0.05% Tyloxapol (Sigma), or on Middlebrook 7H11 agar medium (Difco) supplemented with 10% oleic acid-albumin-dextrose-catalase . Six-to eight-week-old mice were anesthetized with a cocktail of ketamine  and xylasine  and infected intranasally (i.n.) with 1000 CFUs of mycobacteria in 25 μL of PBS containing 0.01% Tween 80. For immunization, C57BL/6 mice were immunized i.n. with 5 × 105 CFU of BCG (Danish) and were challenged 60 days post-vaccination with H37Rv as previously described. All experiments using Mtb were performed in appropriate biosafety level 3 (BSL3) laboratory and animal facility.The laboratory strain of Cell sorted ILC2 were incubated in 6-well plates at a density of 300,000 cells per ml for 4 days with IL-2  and IL-7  in RPMI (Difco) supplemented with 10% FBS. After 4 days of culture, ILC2 were harvested for adoptive transfer.CreERT2 Rosa26tdRFP mice with or without mTOR flox/flox alleles (Id2iΔmTOR) received oral gavage of 5mg Tamoxifen in corn oil four times over the course of 10 days. One week later, mice subsequently received four doses of 100ng rIL-12 and rIL-18 intranasally over a 10-day period, following transient anesthesia with inhaled isoflurane, and were culled to harvest tissues one day after the final dose.Mice were injected intraperitoneally (i.p) one day before infection with either 100 μg of mAb to NK1.1  or 200 μg of mAb to IFN-γ (BioXcell) or its isotype control and the procedure was repeated twice a week until completion of the experiment and sacrifice of the mice. 2-DG  was injected every other day starting from the day of infection and until completion of the experiment. For glucose supplementation, mice were treated with drinking water containing 30% (w/v) glucose (started 1 week before infection until sacrifice). To inducibly delete mTOR in Id2-expressing cells, Id2in vitro cultured of ILC2 were harvested after 7 days of culture and 5x105 to 2x106 cells were transferred i.v. in mice anesthetized with isoflurane one day before Mtb infection in Rag2−/−γc−/−. For the adoptive transfer of the IL-18Rα− or IL-18Rα+ ILC2 subsets, both subsets were FACS-sorted and cultured in vitro for 7 days in complete RPMI supplemented with 10% FBS. At the end of the culture, cells were harvested, and 1 × 105 cells were transferred i.v. in Rag2−/−γc−/− mice anesthetized with isoflurane, one day before i.n. Mtb infection. For ILC1-like transfer, 1x104 purified ILC1-like were directly transferred via intratracheal (i.t.) route in mice anesthetized with isoflurane one day before Mtb infection in Rag2−/−γc−/−.For the adoptive transfer of total lung ILC2, 3, 0.1 mM EDTA (pH 7.2) for immunological staining.Mice were sacrificed any cervical dislocation under isoflurane anesthesia and lungs were harvested aseptically, homogenized using a gentleMACS dissociator  in HBSS (Difco), and incubated with DNAse I  and collagenase D  during 30 min at 37°C under 5% CO2. When indicated, mice received an i.v. injection of labeled anti-CD45 mAb (5μg) 5 min before sacrifice to discriminate between parenchymal and intravascular cells in subsequent flow cytometry analyses. Lung homogenates were filtered on 40 μm cell strainers and centrifuged at 329 × g during 5 min. Supernatants were conserved for cytokine content analysis. Bacterial loads (colony forming units) were determined by plating serial dilutions of the lung homogenates onto 7H10 solid medium (Difco) supplemented with 10% oleic acid-albumin-dextrose-catalase . The plates were incubated at 37°C for 3 weeks before bacterial CFUs scoring. In the remaining fraction, red blood cells were lysed in 150 mM NH4Cl, 10 mM KHCORag2−/− mice were treated intranasally (i.n.) with 100 ng of recombinant IL-33 (Biolegend) each day for 5 consecutive days. For the cytokine-based plasticity model, C57BL/6 or Rag2−/− mice were treated i.n. with different combinations of cytokines specified in figures legends at day 1, 3, 5, 8 and sacrificed at day 9: 100 ng of IL-12 (R&D), IL-18 (R&D), IL-33 (Biolegend) or 20 μg of NMU  per mouse and per instillation.To expand ILC2, C57BL/6 or To identify mouse ILCs, single-cell suspensions were stained with mAb for known lineages and with mAb discriminating ILC subsets. mAbs for known lineages included CD3 , CD4 , CD8a , TCRαβ , TCRγδ,  CD11b , CD11c , F4/80 , Ly6G , TER119 , FcεRIa , CD19 , B220 , and CD49b . mAbs discriminating ILC subsets included CD45.2 , CD90.2 , CD127 , NK1.1 , IL-18Rα , ST2 , CD226 , and CD49a (Ha31/8), NKp46 (29A1.4). mAbs for intracellular staining included GATA3 , T-bet , RORγt , TCF-1 , Arg1 , Ki-67 , and Eomes . After extracellular staining, cells were fixed and permeabilized  for intracellular staining. Samples from Biosafety Level 3 were inactivated for 2 hours at RT with 4% paraformaldehyde (ThermoFisher Scientific) after extracellular and intracellular staining.Live/Dead fixable blue (eBiosciences) and mouse FcBlock (BD Biosciences) were used for all flow cytometry experiments. Cell staining was analyzed using LSR Fortessa flow cytometers (BD) and FlowJo software (v10). Cells were first gated in singlets (FSC-H vs. FSC-W and SSC-H vs. SSC-W) and live cells before further analyses.ex vivo stimulation, cells were incubated in the presence of 10mM 2-DG (Sigma).For intracellular cytokines staining of ILCs, single-cell suspensions from lung were incubated at 37°C with Brefeldin A in association or not with PMA /Ionomycine  or 50 ng/ml of IL-12 and IL-18 for 4 hours before being surface stained, fixed and permeabilized . mAbs for cytokines staining included IFN-γ , IL-17A , IL-5 , and IL-13 . To block glycolysis during −CD45.2+CD90.2+NK1.1−ST2+IL-18Rα+/- ILC1-like were purified as Lin−CD45.2+CD90.2+NK1.1−ST2−CD49a+IL-18Rα+. Cells were sorted using a FACSAria Fusion cytometer .Lung ILCs were enriched from lung single-cell suspensions by using the EasySep™ Mouse ILC2 Enrichment Kit (StemCell). After enrichment, cells were stained with lineage mAb  and ILC markers . ILC2 were purified as Linwww.scenith.com/try-it). Briefly, lung cell suspensions were stimulated for 15 min at 37°C in the presence of the indicated inhibitors of various metabolic pathways then incubated for 30 min with puromycin at 37°C. At the end of the incubation, puromycin was stained with fluorescent anti-puromycin antibodies (Clone R4743L-E8) by flow cytometry and the impact of the various metabolic inhibitors was quantitated as described .Secreted IFN-γ in t test  or Mann-Whitney  were performed when two samples were compared; ANOVA  or Kruskal-Wallis  tests were performed when more than two samples were compared. For all analyses, ∗ indicates p < 0.05, ∗∗ indicates p < 0.01, ∗∗∗ indicates p < 0.001, and ∗∗∗∗ indicates p < 0.0001.Statistical analyses were performed using GraphPad Prism 9 software. Agostino and Pearson normality tests were performed to determine whether data followed a normal distribution. Unpaired


III complex based on 3-pyridylacetic acid and 1,10-phenanthroline ligands shows a dinuclear structure.A novel Dy III complex, [Dy2(C7H6NO2)4Cl2(C12H8N2)2] or [Dy2(μ3-PAA)4(Cl)2(phen)2] , obtained by reaction of Dy(ClO4)3, 3-pyridylacetic acid ligands and 1,10-phenanthroline, exhibits a dinuclear structure. Adjacent binuclear dimers are further connected via face-to-face π–π stacking interactions resulting in supramolecular chains along the c-axis direction.The title Dy Among numerous ligands, pyridinecarboxylate ligands bearing O and N coordination atoms have attracted considerable interest and have proved to be a class of excellent bridging linkers in fabricating metal coordination complexes with appealing structures. The 3-pyridylacetate ligand (3-PAA), one of the most simple pyridinecarboxylate ligands, has attracted particular interest owing to its strong coordination and varied coordination modes, resulting in diverse structures with excellent properties. So far, coordination complexes constructed by the 3-PAA ligand have focused on transition-metal cations, but lanthanide complexes based on the 3-PAA ligand are still rare. Thus, in this work, we prepared the title compound [Dy2(μ2-PAA)4(Cl)2(phen)2] (1) , which displays a dinuclear structure.Coordination complexes composed of metal cations and organic ligands have received much attention because of their diverse structures and intriguing properties such as photoluminescence, magnetism, proton conduction and so on. Lanthanide ions are considered to be excellent metal ions for the construction of such systems because of their unique 41  to 2.5170 (15) Å, and the Dy—N bond distances are 2.5386 (18) and 2.5516 (17)Å, which are similar to those in the complex [Zn(μ-L)(μ-dicl)Dy(NO3)2]·H2O {L = N,N′-dimethyl-N,N′-bisamino] benzene acetate; Echenique-Errandonea et al., 2019− ligands exhibit two different coordination modes. One acts as a tridentate ligand with a μ2-η1:η2 mode, while the other serves as a bidentate ligand with a μ2-κO3:κO4 mode. It is worth emphasizing that the N atom of the PAA ligand is noncoordinating in 1. As shown in Fig. 2III ions are linked by four bridging carboxyl groups of four PAA− ligands, forming the binuclear structure of 1, in which the nearest Dy⋯Dy separation is 3.8976 (19) Å. These adjacent binuclear dimers are further connected via face-to-face π–π stacking interactions involving the phenyl and pyridine rings of the phen ligands, the centroid-to-centroid distance being 3.6116 (10) Å, leading to the formation of supramolecular chains along the c-axis direction 3 (0.2 mmol), 3-pyridylacetic acid , 1,10-phenanthroline (0.25 mmol), HCl (0.25mmol) and Et3N were dissolved in 5 mL of acetonitrile and then sealed into a 25 mL Teflon-lined stainless steel vessel. The vessel was kept at 433 K for 3 d under autogenous pressure and then cooled to room temperature at a rate of 5.63 K h−1. Colorless block-shaped crystals were obtained by filtration of the resulting solution. Yield based on Dy: 38%.Dy I, New_Global_Publ_Block. DOI: 2155088CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


In comparison with the previously reported E-isomer, the reduced planarity of Z-isomer results in a decrease of the contribution of the N+=C(H)—C(H)=B− canonical structure.The title compound has a polarized π-system due to resonance between N—C(H)=C(H)—B and ionic N 20H22BNO2, has a polarized π-system due to resonance between N—C(H)=C(H)—B and ionic N+=C(H)—C(H)=B− canonical structures. The dihedral angles between the ethenyl plane (r.m.s. deviation for C2H2 = 0.0333 Å) with the ethenyl-C(NC2-pyrrole) plane (r.m.s. deviation CNC2 0.0423 Å) and the ethenyl-C plane (r.m.s. deviation BCO2 0.0082 Å) are 45.86 (8) and 37.47 (8)°, respectively, and are greater than those found for the previously reported E-isomer [Hatayama & Okuno —C(H)=B− canonical structure.The title compound, Ckuno 2012 Acta Cry However, the contribution of the ionic canonical structure is very small when p-phenylene is inserted into the N—B bond -2-ethenyl]-9H-carbazole has been reported  and 10.74 (6)° and 5.70 (11) and 9.74 (9)°, respectively —C(H)=B− canonical structure.The dihedral angles between the C13/C14/H13/H14 plane (r.m.s. deviation 0.0333 Å) and the N1/C1/C12/C13 plane (r.m.s. deviation 0.0423 Å) and B1/O1/O2/C14 plane (r.m.s. deviation 0.0082 Å) are 45.86 (8) and 37.47 (8)°, respectively. The relatively large angles result in steric repulsion between carbazolyl and Bpin (pin = pinacolato) residues. The equivalent dihedral angles for the two independent molecules in the In conclusion, structural analyses of both isomers of the hybrid π-system afford an important insight showing the discussed dihedral angles play a crucial role for contribution of the ionic canonical structure.2 , tricyclohexylphosphane  and 4,4,5,5-tetramethyl-1,3,2-dioxaborolane  in a mixture of cyclohexane (10 ml) and triethylamine  was stirred for 3 h under an Ar atmosphere. Powdered 9-ethynyl-9H-carbazole  was added to the solution followed by stirring for 4 h, also under Ar. After a filtration, the filtrate was concentrated under reduced pressure. The residue was extracted with CHCl3, and the solvent was removed via a rotary evaporator. The crude product was purified by gel permeation chromatography (GPC) . 1H NMR : δ 1.05 ; 5.64 ; 7.25 ; 7.41 ; 7.45 ; 7.47 ; 8.04 .A solution of [Rh(cod)Cl]Single crystals of the title compound suitable for X-ray crystallographic analysis were prepared by recrystallization from its hexane solution.Crystal data, data collection and structure refinement details are summarized in Table 110.1107/S2414314621001425/tk4068sup1.cifCrystal structure: contains datablock(s) global, I. DOI: 10.1107/S2414314621001425/tk4068Isup2.hklStructure factors: contains datablock(s) I. DOI: 2061730CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Molecular imaging of CD4+ T cells has potential role for early detection and monitoring of RA. Here, we developed a new radiotracer for in vivo immunoPET imaging of murine CD4+ T cells and tested it in the collagen-induced arthritis (CIA) mouse model of human RA.CD464Cu]Cu-NOTA-CD4-F(ab)’2 ([64Cu]Cu-NOTA-CD4), was generated from F(ab)’2 fragments of R-anti-mouse CD4 antibodies conjugated to the 2-S-(isothiocyanatbenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-SCN-Bn-NOTA) chelator and radiolabeled with copper-64. Accumulation of the tracer and isotype control was evaluated in the CIA model and mice receiving whole-body irradiation (WBI) (5 Gy). The potential of [64Cu]Cu-NOTA-CD4 for response assessment was evaluated in CIA induced mice treated with dexamethasone (DXM). Imaging data were compared with flow cytometry and immunohistochemistry (IHC) of inflammatory cells including CD4+ T cells. [64Cu]Cu-NOTA-CD4 showed increased accumulation in T cell-rich tissues compared with isotype control (p < 0.0001). In addition, reduced accumulation of [64Cu]Cu-NOTA-CD4 was observed in T cell-depleted tissue (p < 0.0001). Flow cytometry and IHC confirmed the increased infiltration of CD4+ T cells in CIA mice.The tracer, [64Cu]Cu-NOTA-CD4, for immunoPET imaging of murine CD4+ T cells. [64Cu]Cu-NOTA-CD4 was successfully synthesized by F(ab)’2 fragments of R-anti-mouse CD4 antibodies conjugated to a chelator and radiolabeled with copper-64. We found that our novel CD4 PET tracer can be used for noninvasive visualization of murine CD4+ T cells.We developed and evaluated a new radiotracer, [The online version contains supplementary material available at 10.1186/s13550-022-00934-7. First, the optimal imaging time point was determined in a longitudinal biodistribution study. Next, the uptake of [64Cu]Cu-NOTA-CD4 following systemic administration of dexamethasone (DXM) was assessed in inflamed arthritic lesions. Finally, target-specific affinity of [64Cu]Cu-NOTA-CD4 and the isotype control radiotracer [64Cu]Cu-NOTA-IgG2b-F(ab)’2 ([64Cu]Cu-NOTA-IgG2b) was evaluated. To validate the findings using other methods, infiltration of CD4+ T cells in the inflamed joints was assessed by flow cytometry and immunohistochemistry (IHC) and compared with CD4 PET imaging.As RA is a T cell-mediated disease, we hypothesized that application of murine T cell-specific antibody F(ab)′2 fragments could be useful to track T cell migration in the CIA model . In the Female DBA/1JRj mice (7 weeks) and C57BL/6JRj (7 weeks) were purchased  and housed in groups of 4–8 mice in individually ventilated cages under standardized lighting conditions. Mice were fed pathogen-free food and water ad libitum. All animal experiments were conducted under protocols approved by the National Animal Experiments Inspectorate under the license number 2016-15-0201-00920.2 and 35% O2 during both injections. Signs of arthritis in the paws appeared approximately four weeks after initial immunization. Each paw was visually scored three times a week on a scale from 0 to 4. The following criteria were used: 0: normal paw, 1: one toe inflamed and swollen, 2: more than one toe, but not entire paw, inflamed and swollen or mild swelling of entire paw, 3: entire paw inflamed and swollen and 4: very inflamed and swollen paw or ankylosed paw Cl2  was dissolved in TraceSelect water (Merck Millipore) to a final concentration of 2 GBq/mL. NOTA-CD4-F(ab)’2 (100 µg in PBS) was incubated with 250 MBq [64Cu]Cl2 in 0.1 M NaoAc buffer pH = 5.5 with 5 mg/ml gentisic acid . The reaction was quenched with 10 mM EDTA followed by PD10 purification into PBS. The radiochemical yield and purity at end-of-synthesis (EOS) were determined by size-exclusion-chromatography-HPLC (SEC-HPLC) using an isocratic method with 0.1 M phosphate buffer pH = 7 as mobile phase and a flow rate of 1 ml/min.[64Cu]Cu-NOTA-CD4 were mixed with NuPAGE LDS sample buffer  and denatured for 10 min at 70 °C and loaded onto BoltTM 4–12% Bis–Tris gels . Electrophoresis was run on the Mini Gel Tank  at 200 V constant voltage in NuPAGE MES SDS running buffer . The SDS-PAGE gel was fixed and stained using Coomassie brilliant blue R-250 . The gel was analyzed for radioactive content by exposure to Multisensitive Phosphor Screens and imaged using the Amersham Typhoon Imaging System .Full-length monoclonal CD4 antibody (IgG), reduced full-length CD4 antibody and the tracer [+ cells were isolated from mouse spleen using the magnetic activating cell sorting (MACS) technique . Female DBA/1JRj mice (7 weeks old) were killed by cervical dislocation and the spleens removed aseptically.Murine CD4+cells (2.5 × 106–4 × 107 cells/mL) were incubated with 1 nM [64Cu]Cu-NOTA-CD4 for 3 h at 4 °C. Cells were centrifuged at 500 g for 5 min and the supernatants and pellets counted in a gamma counter . Cell-associated radioactivity was calculated as the ratio of cell-bound radioactivity to the total amount of added radioactivity.The immunoreactivity of anti-CD4-F(ab)’2 fragments following radiolabeling was assessed according to the Lindmo assay . Increas4 cells) to a MultiScreenHTS BV Filter Plate 1.2 µm  and washed twice in PBS. Eight different concentrations of [64Cu]Cu-NOTA-CD4 [range: 60–0.04 nM] in PBS supplemented with 1% bovine serum albumin (BSA) were added the wells. The plate was incubated for 4 h at 4 °C. After incubation, the plate was washed 3 times in PBS with 1% BSA using a vacuum manifold . The plastic cover was removed from the plate bottom and the plate dried in a heat cabinet. The dry filters were transferred to counting tubes and counted in a gamma counter.The affinity of radiolabeled anti-CD4-F(ab)’2 was assessed by a saturation binding assay. T cells were harvested as described above, added in triplicates (2 × 1064Cu]Cu-NOTA-CD4 and 30 µg CD4-F(ab)’2 co-dose in a total volume of 150 µL (diluted in PBS) in the tail vein as a single bolus. Co-doses was given to decrease binding to lymphoid tissue significantly, but without blocking the specific uptake. The co-dose was based on previously reported data where the optimal dose was found to be 30 µg Cu-NOTA-CD4  and 30 µg CD4-F(ab)’2 co-dose in CIA (n = 6) and control mice (n = 4). For evaluation of DXM, PET/CT imaging was performed 24 h p.i. on day 36 after initial immunization of CIA (n = 23) and DXM-treated mice (n = 12) (four independent experiments: EXP1-4). Untreated control mice (n = 11) were also included. All mice received [64Cu]Cu-NOTA-CD4  and 30 µg CD4-F(ab)’2 co-dose. For isotype control imaging, CIA (n = 8) and control mice (n = 6) were i.v. injected with [64Cu]Cu-NOTA-IgG2b, an isotype-matched non-specific control IgG antibody fragment labeled with 64Cu  and IgG2b-F(ab)’2 30 µg co-dose in a total volume of 150 µL (diluted PBS) in the tail vein as a single bolus. The mice were subjected to PET/CT imaging with 900 s static PET acquisition 24 h p.i. according to the previous described protocol. Whole-body irradiation (WBI) (n = 8/group) and non-irradiated control mice (n = 6/group) were either injected with [64Cu]Cu-NOTA-CD4  or [64Cu]Cu-NOTA-IgG2b  and 30 µg co-dose. PET data were acquired in list mode with an acquisition time of 300, 300, 600 and 1200 s for the 1, 4, 24 and 44 h time points, respectively. PET scans were reconstructed using a maximum a posteriori algorithm with CT-based attenuation correction.Mice were intravenous (i.v.) injected with [be 30 µg . Whole-b64Cu]Cu-NOTA-CD4 and [64Cu]Cu-NOTA-IgG2b was quantified as percent injected dose per joint (%ID/joint) and maximum percent injected dose per gram tissue (%Max ID/g) assuming a soft tissue density of 1 g/cm3. Target-to-blood ratios of [64Cu]Cu-NOTA-CD4 uptake were calculated as maximum uptake (%Max ID/g) in the most affected joints (score 3) divided by mean uptake in the blood (%ID/g) to determine optimal scanning time. Moreover, joint-to-blood ratios were calculated for the DXM assessment.Image analysis was performed using the Inveon® Research Workstation software . A CT-based region of interest (ROI) tool was used to carefully draw around each carpal and tarsal joint, forming four ROIs per mouse. ROIs were also drawn over the heart, kidney, spleen, thymus, liver and thigh muscle. The activity in the blood was calculated as 20% maximum activity in the heart. The uptake of Cu-NOTA-CD4 (n = 8) or [64Cu]Cu-NOTA-IgG2b (n = 8). Two groups of healthy non-irradiated C57BL/6 control mice (n = 6/group) were also included.WBI was performed to test the effect of irradiation on CD4CIA and control mice were euthanized by cervical dislocation and the carpal and tarsal joints were isolated. Joints were decalcified for three weeks in 10% formic acid in buffered formaldehyde 4% followed by preparation in Shandon Excelsior AS Tissue Processor  O/N and embedded in paraffin. Paraffin-embedded joints were sectioned at 4 µM and dewaxed through xylene to tap water. For antigen retrieval, sections were boiled in microwave for 15 min in 10 mmol citrate buffer (pH 6) and pre-incubated in 2% bovine serum albumin (BSA) for 10 min followed by incubation with primary recombinant anti-CD4 antibody  at 1:500 dilution in 2% BSA O/N at 4 °C. Sections were incubated for 40 min with biotinylated secondary goat-anti-rabbit IgG antibody  at 1:200 dilution. Afterward, 3% hydrogen peroxide blocked the endogenous peroxidase. To amplify the reaction, sections were incubated with Avidin and Biotinylated horseradish peroxidase macromolecular Complex (ABC-Elite)  for 30 min. Finally, the reaction was developed by the use of 3,3–diaminobenzidine  for 15 min and counterstaining was performed with Mayer’s Hematoxylin Solution . All procedures were performed at RT if not stated otherwise. Sections were stained in the same analysis. Images were taken using an Olympus BX51 microscope with a XC-10 camera.Joint-infiltrating cells were isolated by a procedure adapted from . BrieflyProcessed cells were blocked with Fc-block (clone 2.4G2)  in FACS buffer (PBS + 0.5% BSA + 0.1% NaN3 + 2 mM EDTA) for 5 min on ice. Fc-blocked samples were stained for 30 min on ice in a master mix of FACS buffer, brilliant stain buffer, amine reactive dye (eFluor780 viability dye)  and the following antibodies: anti-CD3 (clone 145-2C11) PE-CF594, anti-CD4 (clone RM4-5) BV786, anti-CD8 (clone 53–6.7) APC, anti-CD11b (clone M1/70) BV480, anti-CD11c (clone N418) PE-Cy7, anti-CD45 (clone 30-F11) AF700, anti-CD64 (clone X54-5/7.1) BV421, anti-I-A/I-E (clone M5/114.15.2) and anti-Ly6g (clone 1A8) BV711. Antibodies against CD3, CD4, CD8, CD11b, CD45, CD64 and Ly6g were purchased from BD Biosciences. Antibodies against CD11c and I-A/I-E were purchased from BioLegend . Cells were acquired on a BD LSRFortessa X-20 using BD FACSDiva v.8.0.1 software . Data analysis was performed in FlowJo v.10.6.1, Tree Star Inc. .p values < 0.05 were considered statistically significant. Prism 8.0c  was used for all statistical analysis.One-way analysis of variance with Tukey’s post hoc test was used to assess statistically significant differences between groups. 64Cu]Cu-NOTA-CD4 had a high radiochemical purity (> 90%) and aggregates estimated to < 5% by SEC-HPLC. Representative radiochromatogram of [64Cu]Cu-NOTA-CD4 is presented in Fig. 64Cu]Cu-NOTA-CD4 toward CD4 expressing splenocytes was estimated to 90.8% ’2 fragments of R-anti-mouse CD4 antibodies were successfully conjugated to NOTA and radiolabeled with Cu-64 with a radiochemical yield of 79.5 ± 1.5% and a specific activity of 6.5 ± 3.7 MBq/µg. The produced tracer [64Cu]Cu-NOTA-CD4-F(ab)’2 at 100 kDa (lane 3) with no presence of digested fragments or full-length antibody in the final product. In addition, the full-length anti-mouse CD4 antibody (IgG) at 150 kDa (lane 1), the digested antibody (lane 2) with heavy chains (Hc) and light chains (Lc) are shown  followed by a booster immunization with the emulsion in IFA day 21) developed macroscopic joint inflammatory including swelling and erythema of carpal and tarsal joints starting on day 28 after initial immunization. Monitoring of animals was performed on day 28, 32, 33 and 35 with mean CIA score per animal of 0.83 ± 0.24, 1.29 ± 0.16, 1.54 ± 0.18 and 1.63 ± 0.18, respectively  1 h , 4 h  and 24 h  p.i. in comparison with control mice. The uptake in the most pronounced arthritic joints had a drop after the two initial time points in comparison with the two latter time points. At the 44 h p.i. imaging time point the activity was 0.18 ± 0.08% ID/joint. Moreover, the maximum uptake within the most affected joints (score 3) was 15.5 ± 0.9, 16.8 ± 1.8, 11.7 ± 1.3 and 15.9 ± 4.4% ID/g for the 1, 4, 24 and 44 h time points, respectively. The maximum uptake in joints with score 3 was significantly increased at all time points compared with control mice   followed by a decrease at the late time points (24 and 44 h)  Fig. . The act64Cu]Cu-NOTA-CD4 uptake in most affected joints (score 3) significantly increased throughout the imaging time points (1 to 44 h) (p = 0.0071)  was significantly increased in comparison with controls and DXM  (p < 0.0001). The maximum activity within the joints was 9.89 ± 0.78, 10.07 ± 0.93, 6.82 ± 0.65 and 6.10 ± 0.44%ID/g of the CIA mice with score 3, 2, 1 and 0, respectively. Compared with controls and DXM (4.17 ± 0.20% ID/g and 4.21 ± 0.24% ID/g), the maximum activity was significantly increased in all CIA mice  . The CIA mouse shown had score 3 of the right carpus. Joint-to-blood ratios of [64Cu]Cu-NOTA-CD4 uptake 24 h p.i. were significantly higher in CIA mice compared with control  and DXM-treated mice   in comparison with control mice (0.15 ± 0.04% ID/joint and 4.69 ± 0.66% Max ID/g) (p < 0.0001) , thymus (1.58 ± 0.09% ID/g), liver (5.28 ± 0.24% ID/g) and blood (3.84 ± 0.22% ID/g) was calculated. A comparison between [64Cu]Cu-NOTA-CD4 and the [64Cu]Cu-NOTA-IgG2b showed significantly increased activity of [64Cu]Cu-NOTA-CD4 in the spleen (9.77 ± 0.31% ID/g) and thymus (4.65 ± 0.19% ID/g) (p < 0.0001). In addition, isotype activity was significantly increased in the liver (3.28 ± 0.13% ID/g) compared with [64Cu]Cu-NOTA-CD4 (p < 0.0001)  compared with non-irradiated control mice (15.54 ± 0.46% ID/g) (p < 0.0001). This indicates a depletion of CD4+ cells in the spleen following a radiation dose of 5 Gy  compared with DXM (0.08 ± 0.01%). Surprisingly, no significant differences between CIA and control mice were found as results revealed increased CD4+ cell infiltration (0.24 ± 0.02% of viable cells) in the joints of controls  compared with controls (2.2 ± 0.3% of viable cells). In addition, the neutrophil infiltration in joints with score 3 was also significantly increased compared with DXM . Asterisks not shown on graph. Gating strategy, CD45+ T cells and viable cells in joints are shown in Additional file To validate [ols Fig. a. Cell i64Cu]Cu-NOTA-CD4, for immunoPET imaging of murine CD4+ T cells. [64Cu]Cu-NOTA-CD4 was successfully synthesized by F(ab)’2 fragments of R-anti-mouse CD4 antibodies conjugated to a chelator and radiolabeled with copper-64. We found that [64Cu]Cu-NOTA-CD4 could be used for noninvasive visualization of CD4+ T cells. Importantly, [64Cu]Cu-NOTA-CD4 accumulates in T cell-rich tissues compared with the control isotype tracer, [64Cu]Cu-NOTA-IgG2b, and target-specific affinity was also supported by reduced uptake of [64Cu]Cu-NOTA-CD4 in T cell-depleted tissue.In this study, we developed and evaluated a novel radiotracer, [64Cu]Cu-NOTA-CD4 accumulates in tissues with high level of CD4+ T cells and can follow stimuli affected T cell levels. Imaging of CD4+ T cells has an increasingly important role reflecting the pivotal role of CD4+ T cells in many inflammatory diseases including RA, inflammatory bowel disease and multiple sclerosis Zr-malDFO-GK1.5 cDb detected CD4+ T cells in the distal colon of dextran sulfate sodium-induced colitis in a mouse model of inflammatory bowel disease. Beneficially, antibody fragments have shorter circulation times (hours), deeper tissue penetration, allow imaging the same day and can therefore be radiolabeled with shorter-lived PET radionuclides, e.g., flourine-18 (t½ = 109.7 min) and gallium-68 (t½ = 68 min). However, using positron emitters with a longer physical half-lives such as copper-64 (t½ = 12.7 h) enable immunoPET imaging the following day (24 h) Cu-NOTA-CD4 in the spleen and thymus compared with the isotype, [64Cu]Cu-NOTA-IgG2b. For further elucidation, splenic CD4+ T cell depletion was conducted by WBI in mice of 5 Gy. Accordingly, the uptake of [64Cu]Cu-NOTA-CD4 significantly decreased in irradiated mice compared with non-irradiated mice, supporting the target-specific binding of [64Cu]Cu-NOTA-CD4. Similar depletion has previously been shown by Wei et al. Cu-NOTA-CD4 targets CD4+ T cells but also clearly demonstrates the important differences between inflammatory and healthy tissues including vascular changes, increased blood flow, poor lymphatic drainage and edema Cu-NOTA-IgG2b, in joints with pronounced arthritis. No increased uptake in tissues with mild arthritis and healthy tissue was observed, indicating specific targeting of our CD4 PET tracer, [64Cu]Cu-NOTA-CD4, in lesions with mild inflammation. Therefore, we speculate that the specificity of our CD4 PET tracer toward the increased number of CD4+ cells is sufficient to exceed the passive targeting by the EPR effect or potentially uptake by phagocytosis in joints with mild inflammation. Kristensen et al. Zr-DFO-CD4 and [89Zr]Zr-DFO-CD8a, in several preclinical mouse models of cancer. Here, tracers were used to phenotype tumors at an early stage and also allow following the treatment response. The possibility of unspecific tracer uptake in the heterogenic tumor microenvironment caused by the EPR effect was also discussed. Careful assessment and inclusion of isotype controls are therefore of great importance for indications beyond inflammation. Altogether, this indicates that the use of our CD4 PET tracer, [64Cu]Cu-NOTA-CD4, should be centered toward following the response to treatment and detecting potential recurrence rather than initial diagnosis. It could be argued that imaging of inflammation could easier be done with [18F]-FDG PET. However, it has previously been shown that not only is the uptake of [18F]-FDG low in arthritis but also that it seems not to correlate with severity of arthritis, i.e., arthritis score Cu-NOTA-CD4, for immunoPET imaging of murine CD4+ T cells. [64Cu]Cu-NOTA-CD4 was successfully synthesized by F(ab)’2 fragments of R-anti-mouse CD4 antibodies conjugated to a chelator and radiolabeled with copper-64. We found that our novel CD4 PET tracer can be used for noninvasive visualization of murine CD4+ T cells and may be used for noninvasive studies of inflammatory conditions including RA.We developed and evaluated a novel radiotracer, [Additional file 1: Figure S1 Full-length SDS-PAGE. (a) Coomassie staining of a full-length SDS-PAGE with ladder. (b) radiography of the same full-length stained SDS-PAGE. Figure S2 Gating strategy used to identify CD4+ T cells and neutrophils by flow cytometry. Percent of (a) CD45+ T cells and (b) viable cells in the carpal and tarsal joints from control, DXM-treated and untreated CIA mice. Mice with CIA were scored according to degree of inflammation (swelling and erythema) in each paw on a scale from 0–4. Each point represents a joint. Data are pooled from two independent experiments. The significance level is indicated by asterisks (*). p = 0.02–0.04 (*) and p < 0.0001 (****). (c) Both populations were gated based on time, as singlets, based on scatter profile, lack of staining by viability dye and as positive for CD45. CD4+ T cells were further defined as CD3+ CD4+. Neutrophils were further defined as CD11b+ Ly6g+.


E)-2-methyl-4-(quinolin-8-yldiazenyl)benzene-1,3-diol ligand was prepared and structurally characterized. The UV–Vis absorption spectra of the ligand and the complex are reported.A copper(II) complex with the ( E)-2-methyl-4-(quinolin-8-yldiazenyl)benzene-1,3-diol ligand in acetonitrile leads to the formation of the title compound, [Cu(C16H12N3O2)Cl]·H2O. The ligand is deprotonated and coordinates with three donor atoms (tridentate) to the CuII ion. Individual molecules of the CuII complex are connected by chloride bridges, forming a one-dimensional coordination polymer. No photoisomerization to the cis isomer of the azo ligand was observed upon irradiation with UV light.The reaction between copper(II) chloride dihydrate and the ( The combination of azo compounds with metal ions to form complexes is a promising approach for controlling their photophysical properties. In metal complexes with azo ligands, the metal centers and azo ligands can affect each other’s properties. For example, cis–trans photoisomerization by irradiation with a single frequency of light has been achieved in azo-conjugated metal complexes by a combination of the photophysical and the redox properties of ligand and metal center -2-methyl-4-(quinolin-8-yldiazenyl)benzene-1,3-diol, to investigate azo–hydrazone tautomerism in its CuII complex. The photophysical properties of the ligand and the CuII complex were studied by UV–Vis spectroscopy to address the potential photoisomerization.Azobenzene derivatives are well-known dyes with fascinating characteristics such as 2.II complex is shown in Fig. 1II complex and one solvent water molecule. The hydroxy group in the ortho-position of the azo ligand is deprotonated and is coordinated the CuII center. In the asymmetric unit, the CuII ion is 4-coordinated in a distorted square-planar geometry. The donor atoms comprise one nitrogen atom of the quinoline moiety, one nitrogen atom of the azo group, one deprotonated alcohol oxygen atom, and a chloride ion. The other hydroxy group of the azo ligand, in the para-position, remains protonated. The chlorido ligand is also weakly coordinated by an adjacent CuII center occupying its apical position, resulting in an elongated square-pyramidal coordination polyhedron around the copper(II) ions. The Cu1—Cl1i distance in the apical position is 2.7395 (10) Å, which is notably longer than the distances in the equatorial positions, Cu1—Cl1 = 2.2803 (8) Å, Cu1—O1 = 1.917 (2) Å, Cu1—N1 = 2.008 (3) Å, and Cu1—N2 = 1.945 (3) Å . The N2—N3 bond distance of 1.293 (4) Å is typical for the N=N double bond of an azo group. The structural features of the aromatic rings and the C11—O1 single-bond length of 1.300 (4) Å also indicate that the ligand adopts the azo structure, rather than the hydrazone structure, which is similar to the structures observed in analogous azo-metal complexes with other metals, including Ni, Cu, and Zn  x + y + z − x − y + z − The coordinated chloride ion bridges adjacent Cuon Fig. 2. This is] Table 1.4.et al., 2016ConQuest  was observed at 406 nm for the ligand, while the CuII complex showed decreased absorption and red-shifted maxima at 420 and 489 nm. To investigate the photoisomerization of the ligand and the CuII complex, the samples were irradiated at maximum wavelength, but no photoisomerization to the cis isomer was observed for either compound.The UV–Vis spectra of the azo ligand and the Cu6.M NaNO2 (3 mL) was slowly added to a cold solution of 8-aminoquinoline  in 0.5 M HCl(aq) (20 mL). The resulting solution was stirred at 277 K for 15 min, and an aqueous solution of (NH2)2CO  in 3 mL of water was then added to give a diazonium chloride solution. This solution was added to an aqueous 0.25 M NaOH solution of 2,6-dihydroxytoluene  and stirred at 277 K for 30 min and then stirred at room temperature for 15 h. The reaction mixture was acidified with 1 M HCl(aq) (10 mL) and a red precipitate was formed. The precipitate was filtered off and washed with water and then with cold tetrahydrofuran. Yield, 86% . IR: νmax (KBr): 3400, 3068, 1633, 1536, 1503, 1488, 1447, 1364, 1299, 1212, 787 cm−1. 1H NMR : δH 9.06 , 8.41 , 8.20 , 7.97 , 7.74 , 7.62 , 7.49 , 6.63 , 2.12 . Analysis calculated for C16H13N3O2·0.72HCl: C, 62.90; H, 4.53; N, 13.75. Found: C, 62.49; H, 4.31; N, 14.17. The CuII complex was obtained as a brown solid by the reaction of the azo ligand synthesized as described above  in 4 mL of ethanol with CuCl2·2H2O  in 2 mL of H2O. Yield, 54% . Crystals of the CuII complex suitable for the X-ray crystallography study were obtained by the slow diffusion of a CH3CN solution of the ligand into an aqueous solution of CuCl2·2H2O. IR: νmax (KBr): 3418, 2924, 2854, 1633, 1557, 1508, 1436, 1283, 1258, 1048 cm−1. Analysis calculated for C16H12ClCuN3O2: C, 50.94; H, 3.21; N, 11.14. Found: C, 50.82; H, 3.63; N, 11.49.To synthesize the title ligand, an aqueous solution of 1.2 7.para-position were found in the difference-Fourier map and were refined isotropically without restraints or constraints. Other hydrogen atoms were generated geometrically, and refined with a riding model with C—H = 0.98 Å, Uiso(H) = 1.5 Ueq(C) for methyl, and C—H = 0.95 Å, Uiso(H) = 1.2 Ueq(C) for aromatic hydrogen atoms. Two reflections were omitted as clear outliers.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989022003437/yz2018sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989022003437/yz2018Isup2.hklStructure factors: contains datablock(s) I. DOI: 2162331CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Escherichia coli O125, the cause of infectious diarrheal disease, is comprised of two serogroups, viz., O125ab and O125ac, which display the aggregative adherence pattern with epithelial cells. Herein, the structure of the O-antigen polysaccharide from E. coli O125ac:H6 has been elucidated. Sugar analysis revealed the presence of fucose, mannose, galactose and N-acetyl-galactosamine as major components. Unassigned 1H and 13C NMR data from one- and two-dimensional NMR experiments of the O125ac O-antigen in conjunction with sugar components were used as input to the CASPER program, which can determine polysaccharide structure in a fully automated way, and resulted in the following branched pentasaccharide structure of the repeating unit: →4)[β-d-Galp-(1 → 3)]-β-d-GalpNAc-(1 → 2)-α-d-Manp-(1 → 3)-α-l-Fucp-(1 → 3)-α-d-GalpNAc-(1→, where the side chain is denoted by square brackets. The proposed O-antigen structure was confirmed by 1H and 13C NMR chemical shift assignments and determination of interresidue connectivities. Based on this structure, that of the O125ab O-antigen, which consists of hexasaccharide repeating units with an additional glucosyl group, was possible to establish in a semi-automated fashion by CASPER. The putative existence of gnu and gne in the gene clusters of the O125 serogroups is manifested by N-acetyl-d-galactosamine residues as the initial sugar residue of the biological repeating unit as well as within the repeating unit. The close similarity between O-antigen structures is consistent with the presence of two subgroups in the E. coli O125 serogroup.Enteropathogenic  Escherichia coli is the most abundant of the facultatively anaerobic microbes in the gastrointestinal tract of mammals, colonizing human infants within hours of birth  and flagellar (H) antigens, and several of these are causative agents of diarrhea, cystitis, pyelonephritis and meningitis (E. coli (EPEC) are associated with severe infections of the gastrointestinal tract in humans. The O125 serogroup, originally identified during a diarrheal outbreak in London in 1952 . Sugar analysis of the E. coli O125ab and O125ac PS revealed fucose, mannose, glucose, galactose, galactosamine ; the 1H and 13C NMR chemical shifts obtained herein correlated well with the previously published data   and SWppm is the spectral width in ppm   set to 2f 16.4 T . In bothh in ppm . In the h in ppm . In bothh in ppm . The peaE. coli O125ac PS, the magnitude of 3JH1,H2 of anomeric protons in the 1D 1H NMR spectrum  and chemical shifts from the 13C NMR spectrum were entered. From 2D NMR data correlations observed in 1H,1H-TOCSY, 1H,13C-HSQC, 1H,13C-H2BC, 1H,13C-HMBC spectra were used as input to the program (Supporting information) as well as 1JC1,H1 of anomeric nuclei  from an F2-coupled 1H,13C-HSQC spectrum. Additional input that can be provided is information on sugar components, and if the Wzx/Wzy biosynthetic pathway can be anticipated . This “biological rule” related to WecA should pertain to the E. coli O125ac O-antigen possessing four different sugar residues in its RU as seen from the sugar analysis. The NMR data from the above described experiments, the “activated” WecA rule in conjunction with l-Fuc, d-Man, d-Gal and two d-GalNAc as monosaccharide residues, along with all possible linkages available were used as input to the CASPER program using the module “determine glycan structure”.Determination of the polysaccharide structure using sugar components and unassigned NMR data can be carried out by the computer program CASPER  as shownicipated . The latd-Glc residue. The assigned experimental 1H and 13C NMR chemical shifts (vide infra) correlated quite well with the chemical shifts predicted by CASPER -β-d-GalpNAc- indicating spin-systems from residues having the galacto-configuration. Arising from D1 and E1 two correlations were observed for each sugar residue when the shortest mixing time of 30 ms was used, viz., to δH 4.20 and 3.98 and to δH 3.54 and 3.63, respectively; in each of the spin systems, a third cross-peak was observed to δH 4.29 (D4) and to δH 3.93 (E4) when mixing times in the range 60–200 ms were used. The poorer transfer of magnetization in B1/C1 compared to D1/E1 is consistent with the former having the α-anomeric configuration and the latter, the β-anomeric configuration, in complete agreement with the results from 1JC1,H1 coupling constants of the sugar residues. This analysis also confirms the lack of a sugar residue having the gluco-configuration in the RU, and consequently the O125ac PS is devoid of a glucosyl residue in its O-antigen.In 1H,13C-HSQC, 1H,13C-H2BC, 1H,13C-HMBC and 1H,13C-HSQC-TOCSY experiments; the two experiments with TOCSY transfer employed mixing times of 20 and 120 ms, respectively. Starting from A1 correlations were observed to δH/δC 4.21/77.71 (A2) via cross-peaks in both 1H,13C-H2BC and 1H,13C-HSQC-TOCSY spectra and further from A2 cross-peaks throughout the spin-system were observed due to large 3JH,H coupling constants from H2 and onward in mannosyl residues; combined with information form 1H,13C-H2BC spectra resonances from A3–A6 could thus be fully assigned.The spin-systems were thereafter fully assigned using a combination of δH/δC 1.22/16.05 (B6) with subsequent correlations observed in the 1H,13C-H2BC and 1H,13C-HSQC-TOCSY spectra to δH/δC 4.18/67.88 (B5) as well as a 1H,13C-HMBC correlation due to 3JH6,C4 observed at δC 72.42 corresponding to B4. From anomeric positions C1 and D1 both display cross-peaks in the 1H,13C-H2BC spectrum to δH/δC 4.44/49.85 (C2) and δH/δC 4.20/52.63 (D2), respectively, the 13C NMR chemical shifts of which are characteristic of 2-amino-2-deoxy-sugars  spectra facilitated chemical shift assignments for the hydroxymethyl groups of residues A, C, D and E in both polysaccharides as well as the one in residue G of the O125ab PS to be fully assigned  torsion angle of the branched →3,4)-β-d-GalpNAc-, 3.79/3.81/60.80 (C), 3.72/3.82/61.09 (D), 3.77/3.79/61.98 (E) and 3.75/3.88/61.87 (G).The high resolution in the assigned . Interesus study  and are 1H,13C-HMBC and 1H,1H-NOESY spectra -α-d-Manp-(1 → 3)-α-l-Fucp-(1 → (A-B). In a polysaccharide model of the O125ac PS generated by CarbBuilder  showed, besides from the expected trans-glycosidic linkage correlation, G1 to A3, an inter-residual correlation between A2 and G5  showed that G1-A3 < G1-G2 < G5-A2. Based on molecular model-derived proton-proton distances the structural element α-d-Glcp-(1 → 3)-α-d-Manp--β-d-Manp-, i.e. it was present at δH/δC 4.47/105.50 presumed to originate from a similar sugar residue denoted by E1’. The NMR chemical shifts of the low intensity cross-peak are in excellent agreement with predictions made by CASPER for the β-d-Galp-(1 → 3)-β-d-GalpNAc disaccharide structural element, i.e. δH/δC 4.48/105.50, thereby lending support to that this H1/C1 correlation in the spectrum arises from the terminal sugar residue in the O-antigen, consistent with a d-GalpNAc residue at the reducing end of the biological repeating unit (BRU). Furthermore, the number of RUs of the O-antigen polysaccharide was estimated by integration of the 13C resonances from the anomeric carbons of residues E1 and E1’ and was found to be ~15.In the manBC is consistent with the mannosyl residue in the polysaccharides. Furthermore, in the Wzx/Wzy biosynthetic pathway of O-antigen polysaccharides   . While oP-Glc precursor in the cytoplasm. This is followed by the flipping of Und-P-Glc into the periplasm by GtrA and subsequent transfer of the glucosyl group by a specific glucosyl transferase (Gtr) to the acceptor molecule. Notably, this sequence is similar to the glucosylation that occurs in modification of e.g. the Shigella flexneri serotype 2a O-antigen (d-Glcp-(1 → 3)-linked to RhaI of the RU, resulting in branched structures within the O-antigen.Additionally, we speculate that the glucosyl group in the O125ab O-antigen is added at the periplasmic side of the inner membrane in a three-step process  whereby -antigen , in whicE. coli O125ab and O125ac  and O125ab (E. coli O125ab:H19) were obtained from SSI Diagnostica A/S . Bacterial culture, LPS extraction and preparation of lipid-free polysaccharides (PS) were carried out as previously described (escribed .4 in aq. ammonia (1 M) at ambient temp for 30 min and acetylation at 100 °C for 30 min using Ac2O and pyridine (1:1). The resulting mixtures of alditol acetates were analyzed by gas chromatography–mass spectrometry (GC–MS) and separated on an HP-5MS column attached to a Shimadzu GCMS-QP2020 with an electron impact (EI) source. The temperature program used an injection temperature of 250 °C and an initial oven temperature of 60 °C with a hold time of 2 min, followed by ramped heat increase @ 20 °C min−1 to 200 °C then 5 °C min−1 to 260 °C with a hold time of 4 min at 260 °C. The samples were compared with authentic standard references and their mass fragments. The retention times of the derivatives were compared to those of authentic standards as references and their mass spectra were compared to EI-MS fragmentation patterns (Polysaccharides purified by gel-filtration chromatography (PS) from O125ab and O125ac were hydrolyzed with 2 M TFA at 120 °C for 30 min followed by reduction with NaBHpatterns .2O and examined by NMR spectroscopy as solutions in 99.96 percent D2O (8 mg in 0.55 mL) with a trace amount of NaN3 prior to 1D 13C NMR, 2D 1H,13C-HMBC, 1H,13C-H2BC and 1H,13C-BS-CT-HMBC experiments. Samples of O125ab and O125ac PS purified by gel-permeation chromatography were used for additional NMR experiments (1 mg in 0.55 mL). NMR spectra were recorded at 70 °C using Bruker Avance 500 MHz or Bruker Avance III 700 MHz spectrometers equipped with 5 mm TCI (1H/13C/15N) Z-Gradient (53.0 G cm−1) CryoProbes. Chemical shifts are reported in ppm using internal sodium 3-trimethylsilylpropanoate-2,2,3,3-d4  for 1H NMR and external 1,4-dioxane 10 percent in D2O (δC 67.40) for 13C NMR as references. Chemical shift differences were obtained by comparison to NMR data of the corresponding monosaccharides  by Iguchi et al. (KP835694 for O125ab and KP835695 for O125ac) by DebRoy et al.  and the Knut and Alice Wallenberg Foundation to G.W. K.U. acknowledges support from the Swedish Research Council (2012-03564).Conflict of interest statement: None declared.Supporting_information_O125_GC-MS_cwac061Click here for additional data file.Supporting_information_O125_NMR_220724_cwac061Click here for additional data file.


Two organic salts based on 1,3,4 thiadiazole derivatives have been obtained and their structures have been established by single-crystal X-ray analysis. 4H8N3S+·0.5C2O42−, (I), and 4-(dimethylamino)pyridin-1-ium 4-phenyl-5-sulfanylidene-4,5-dihydro-1,3,4-thiadiazole-2-thiolate, C7H11N2+·C8H5N2S3−, (II). Both solids were investigated by single-crystal X-ray diffraction and by Hirshfeld surface analysis. An infinite one-dimensional chain along [100] is generated through O—H⋯O interactions between the oxalate anion and two 2-amino-5-ethyl-1,3,4-thiadiazol-3-ium cations in compound (I), and a three-dimensional supramolecular framework is generated through C—H⋯O and π–π interactions. In compound (II), an organic salt is formed by a 4-phenyl-5-sulfanylidene-4,5-dihydro-1,3,4-thiadiazole-2-thiolate anion and a 4-(dimethylamino)pyridin-1-ium cation, which are combined by an N—H⋯S hydrogen-bonding interaction, forming a zero-dimensional structural unit. As a result of intermolecular π–π interactions, the structural units are combined into a one-dimensional chain running along the a-axis direction.During attempts to achieve interaction between 2-amino-5-ethyl-1,3,4-thiadiazole with oxalyl chloride and 5-mercapto-3-phenyl-1,3,4-thiadiazol-2-thione with various diacid anhydrides, we obtained two co-crystals , namely, 2-amino-5-ethyl-1,3,4-thiadiazol-3-ium hemioxalate, C Heterocyclic compounds with the 1,3,4-thiadiazole structural unit are very attractive for the production of pharmaceuticals as 1,3,4-thiadiazole derivatives exhibit a wide spectrum of biological activities. The 1,3,4-thiadiazole moiety acts as a hydrogen-binding dominant unit on the one hand and as an electron-donor unit on the other (Sharma 2.The molecular structure of compound (I)3.i.e. the molecular ratio is 2:1. The oxygen atoms of the oxalate anion are involved in intermolecular hydrogen bonding  = 3.7734 (10) Å, where Cg1 is the centroid of the S1/C1/N2/N3/C2 ring].In the asymmetric unit of compound (I)g Table 1 with neig Table 1. Each ch al. 1993. These cO Table 1 and π–π i.e. the molecular ratio is 1:1. The cation and anion are combined by an N—H⋯S hydrogen-bonding interaction  = 4.311 (2) Å, Cg1 is the centroid of the C3–C8 ring], the structural units combine as a building block of a one-dimensional chain running along the a-axis direction n Table 2 and formon Fig. 5.4.et al., 2016et al., 1999A/N21A site to the two carboxylate oxygen atoms. The second structure is for bis(4-aminopyridine-N)trimethyltin with 3-phenyl-1,3,4-thiadiazoline-2-thione-5-thiolate  Å, 159°] with the 1,3,4-thiadiazole molecule.A search of the Cambridge Structural Database rm Fig. 6 shows thrm Fig. 6 reveal trm Fig. 6.6.Synthesis of 2-amino-5-ethyl-1,3,4-thiadiazole:−1): 3290, 2980, 2780; 1640.Propionic acid (0.108 mol) was mixed with 16 g of sulfuric acid (94%). The reaction temperature was allowed to reach 333–343 K, and then, under the same conditions, 0.1 mol of thiosemicarbazide were added. The mixture was stirred for 3 h at 333–343 K, water and charcoal were added, and the mixture was stirred for 40 minutes. At the end of the reaction, the solution was filtered. Then, 44% sodium hydroxide solution was added to get a solution with pH 9.5–10. After cooling the reaction to 303–308 K, the mixture was filtered. The precipitate was washed with water (303 K) and allowed to dry to give the title compound , m.p. 460–467 K. IR  al. 1984. We trieet al., 2010Compound (II)7.Uiso(H) = 1.2Ueq or 1.5Ueq(C-methyl)]. In (II)Uiso(H) = 1.5Ueq(C)].Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989022012154/dj2057sup1.cifCrystal structure: contains datablock(s) I, II, global. DOI: 10.1107/S2056989022012154/dj2057Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989022012154/dj2057IIsup3.hklStructure factors: contains datablock(s) II. DOI: 2232672, 2232671CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information: