Brown adipose tissue (BAT) burns fatty acids (FAs) to produce heat, and shows diurnal oscillation in glucose and triglyceride (TG)-derived FA-uptake, peaking around wakening. Here we aimed to gain insight in the diurnal regulation of metabolic BAT activity.RNA-sequencing, chromatin immunoprecipitation (ChIP)-sequencing, and lipidomics analyses were performed on BAT samples of wild type C57BL/6J mice collected at 3-hour intervals throughout the day. Knockout and overexpression models were used to study causal relationships in diurnal lipid handling by BAT.Lpl) expression, and was predicted to be driven by peroxisome proliferator-activated receptor gamma (PPARγ) activity. ChIP-sequencing for PPARγ confirmed oscillation in binding of PPARγ to Lpl. Of the known LPL-modulators, angiopoietin-like 4 (Angptl4) showed the largest diurnal amplitude opposite to Lpl, and both Angptl4 knockout and overexpression attenuated oscillations of LPL activity and TG-derived FA-uptake by BAT.We identified pronounced enrichment of oscillating genes involved in extracellular lipolysis in BAT, accompanied by oscillations of FA and monoacylglycerol content. This coincided with peak lipoprotein lipase (Our findings highlight involvement of PPARγ and a crucial role of ANGPTL4 in mediating the diurnal oscillation of TG-derived FA-uptake by BAT, and imply that time of day is essential when targeting LPL activity in BAT to improve metabolic health.  •The transcriptome and lipidome of brown fat show clusters with distinct circadian phases.•The peak in metabolic brown fat activity is defined by activation of lipolytic processes.•PPARγ shows oscillating binding to lipolytic genes and may drive diurnal brown fat activity.•Angptl4 flattens rhythmic activity in brown fat.Genetic modulation of the lipoprotein lipase inhibitor •Time of day should be considered when studying the metabolic benefits of targeting brown fat. ABCD1ATP binding cassette subfamily D member 1ACACAAcetyl-CoA carboxylaseACLYATP citrate lyaseACSL1Acyl-CoA synthetase long chain family member 1ANGPTLAngiopoietin-likeAPOApolipoproteinBATBrown adipose tissueCD36Cluster of differentiation 36CEBPACCAAT/enhancer-binding protein alphaCEBPBCCAAT/enhancer-binding protein betaCES1DCarboxylesterase 1 DChEA3ChIP-X enrichment analysis version 3CIDECCell death inducing DFFA like effector CCLOCKCircadian locomotor output cycles kaputDBPNuclear receptor subfamily 1 group D memberDGDiacylglycerolDGAT2Diacylglycerol O-acyltransferase 2ELOVLElongation of very long chain fatty acids proteinFAFatty acidFASNFatty acid synthaseGAPDHGlyceraldehyde 3-phosphate dehydrogenaseGCKGlucokinaseGPAT4Glycerol-3-phosphate acyltransferase 4GPRG-protein-coupled receptorGPIHBP1Glycosylphosphatidylinositol anchored high density lipoprotein binding protein 1GTF2IGeneral transcription factor II-IHACD43-hydroxyacyl-CoA dehydratase 4HDLHigh-density lipoproteinHRMSHigh-resolution mass spectrometryiBATInterscapular brown adipose tissueKOKnockoutLPLLipoprotein lipaseMGMonoacylglycerolMT-CYTBMitochondrial cytochrome bMT-NDNADH-ubiquinone oxidoreductase chainNOCTNocturninPLIN4Perilipin 4PLTPPhospholipid transfer proteinPNPLA2Patatin like phospholipase domain containing 2PPARγPeroxisome proliferator-activated receptor gammaPUFAPoly-unsaturated fatty acidqPCRQuantitative polymerase chain reactionRPLP060S acidic ribosomal protein P0sBATSubscapular brown adipose tissueSCD1Stearoyl-CoA desaturase-1SREBF1Sterol regulatory element-binding transcription factor 1TECRTrans-2,3-enoyl-CoA reductaseTGTriglycerideTgTransgenic overexpressionTRLTriglyceride-rich lipoproteinUCP1Uncoupling protein 1UPLCUltra-performance liquid chromatographyWATWhite adipose tissueWTWild typeZTZeitgeber time1White adipose tissue (WAT) is the most abundant type of adipose tissue that humans and other mammals have, which primarily stores energy from food. In contrast, the main function of brown adipose tissue (BAT) is to convert energy into heat, a process known as non-shivering thermogenesis. These two types are intertwined and highly related to one another as for example white adipocytes are capable of transdifferentiating into so-called beige adipocytes with brown-like characteristics. Brown and beige adipocytes produce heat via expression of uncoupling protein 1 (UCP1), which dissipates the mitochondrial proton motive force generated by fatty acid (FA) oxidation thereby releasing energy as heat . ConsequUcp1 expression during the wakeful (active) phase [Nr1d1 expression (encoding REV-ERBA) to relieve the inhibition on Ucp1 expression [de novo lipogenesis with peak expression near the end of the feeding (active) phase of mice, which probably serves to provide BAT with extra fuel to maintain body temperature during the resting (inactive) phase and/or for combustion towards the start of the subsequent wakeful phase.We and others have shown that nutrient uptake by BAT is characterized by strong diurnal oscillations, with peak uptake of glucose and TG-derived FAs around wakening . This die) phase . Interespression . Similarpression  recentlyAngptl)4 as critical mediator in the diurnal regulation of lipoprotein lipase (LPL) activity, thereby governing the diurnal regulation of TG-derived FA-uptake by BAT.Here we aimed to gain further insight into the diurnal regulation of metabolic BAT activity. To this end we used an unbiased RNA sequencing and lipidomics approach in BAT of mice, and identified angiopoietin-like (22.1Zeitgeber Time (ZT) where ZT0 indicates the onset of the light (inactive) phase. Oscillation of transcripts was assessed by JTK cycle analysis, which provided the phase, amplitude, and significance values for each transcript and showed that in total, 5,486 genes  (full details are provided in RNA-sequencing was performed on interscapular BAT (iBAT) samples collected at 3-hour intervals throughout a 24-hour period from chow-fed male C57BL/6J mice exposed to mild cold . All miP = 7.77 · 10−29)  showed peak expression around ZT18 B and was· 10−29) B. These P = 4.56 · 10−7)  peaked around the onset of the light phase at ZT0 B, and wa · 10−7) B. This ide novo lipogenesis were identified (Acly) and acetyl-CoA carboxylase (Acaca). Glucokinase (Gck), another gene involved in de novo lipogenesis, displayed oscillation with a slightly earlier peak expression at ZT18 corresponding to cluster 1. Despite the sub-thermoneutral housing temperature of 22 °C, no oscillations were identified for FA synthase (Fasn), stearoyl-CoA desaturase-1 (Scd1), and elongation of very long chain FAs protein (Elovl)3  as a potential mediator of the gene expression in clusters 1 and 2 B. GTF2I entified  in line (Elovl)3 . This inP = 7.92 · 10−7; Cluster 3  had a peak expression around ZT8 B, and waP = 2.31·10−6)  was identified with peak expression around ZT14 B, coinci31·10−6) B.Cebpa expression by itself was found oscillating within cluster 3, while expression of Pparg itself and Noct were not oscillating (data not shown), likely because PPARγ is broadly regulated at the post-transcriptional level [de novo lipogenesis [Transcription factor enrichment analysis revealed peroxisome proliferator-activated receptor gamma (PPARγ) as the top hit for both cluster 3 and 4. PPARγ senses the energy status of the cell to regulate lipid uptake and storage, and in fact has often been described as an important circadian transcription factor in adipose tissue ,27. Its al level . Strikinogenesis  and is rogenesis . Collecti.e., around ZT12), relative amplitudes of individual genes were compared within clusters 3 and 4 (Nr1d1) and D-box binding protein (Dbp), both involved in the core clock machinery, as the genes with largest relative amplitude in cluster 3 and 4, respectively. The genes that follow in cluster 3 are involved in a broad variety of cellular processes including cytokine binding and amino acid metabolism, while the three genes that follow in cluster 4 are all involved in the cellular core clock machinery.To obtain further insight in the processes that take place during the time that BAT is metabolically most active ( 3 and 4 . We idenmt-nd1), a subunit of NADH dehydrogenase critical for the electron transport chain, was identified as the gene with the highest diurnal amplitude within the third cluster, with estimated peak expression near ZT8. The four genes that follow within this cluster encode for mitochondrial complexes and Ucp1, suggesting that large diurnal changes in mitochondrial dynamics are required for the flexible regulation of FA combustion [Although of interest, these data do not explain the diurnal metabolic activity in BAT, and we therefore proceeded with comparing absolute amplitudes of individual genes . NADH-ubmbustion .Lpl, encoding for the protein responsible for liberating FAs from TRLs [i.e. glycerol-3-phosphate acyltransferase 4 (Gpat4), diacylglycerol O-acyltransferase 2 (Dgat2), cell death inducing DFFA like effector C (Cidec), and Perilipin 4 (Plin4)) and intracellular lipolysis (i.e. acyl-CoA synthetase long chain family member 1 (Acsl1), patatin like phospholipase domain containing 2 (Pnpla2), and carboxylesterase 1 D (Ces1d)).Interestingly, we identified a large absolute diurnal amplitude for rom TRLs , in clusi.e. PPARγ binding sites), 8,153 demonstrated diurnal oscillation as determined by JTK-cycle analysis , with estimated peaks in PPARγ binding on average 2 h prior to estimated peak in gene expression -sequencing for PPARγ on pooled iBAT samples. Out of 66,066 peaks  phase serves to supply BAT with fuel for thermogenesis. This seems to be followed by storage of FA taken up from the circulation after hydrolysis of TRLs by LPL and subsequent lipogenesis around the onset of the dark (active) phase, likely in order to replenish intracellular lipid stores. PPARγ likely mediates as a driving factor in the transcriptional control of genes involved in the uptake, storage, and intracellular lipolysis of lipids.2.2i.e. sample) clustering is presented in Abcd1), Elovl5, 3-hydroxyacyl-CoA dehydratase 4 (Hacd4), and trans-2,3-enoyl-CoA reductase (Tecr) showed peak expression throughout the light phase -high-resolution mass spectrometry (HRMS)-based lipidomics on the same iBAT samples. Out of 1,941 measured lipid species, 430 demonstrated diurnal oscillation as analyzed by JTK A full d. For 11 ht phase .Figure 3de novo lipogenesis, and metabolically inactive BAT.Cluster 2 (peak ZT21) contained the remaining oscillating DGs, characterized by primarily short or medium acyl chains (<C18:x). In addition, this cluster contained 67 of the 74 oscillating TGs B, probabCluster 3 (peak ZT16) contained 11 of the 13 oscillating FAs and eight of the nine oscillating monoacylglycerols MGs; B. These 2.3Lpl as the gene with the largest amplitude within this cluster, highlighting its relevance in the diurnal oscillation of TG-derived FA-uptake by BAT [Angptl3, Angptl4, and Angptl8, apolipoprotein (Apo)c1, Apoc2, Apoc3, Apoa5 [Gpihbp1), FA transporter cluster of differentiation 36 (Cd36), and phospholipid transfer protein (Pltp). Strikingly, besides the strong oscillation of Lpl expression  and lowest (ZT15) measured levels opposing the oscillation of Lpl expression . Similarly, gene expression of Gpihbp1, which protects LPL from catalysis by ANGPTLs, and the FA transporter Cd36 showed no oscillation (Pltp (We observed peak expression of genes involved in lipid catabolic processes around ZT12 (onset of the dark phase) and identified e by BAT . To deli3, Apoa5 , and relpression A, expresillating B–D. In ppression B. It wasur delay , illustrur delay . ANGPTL8ur delay , therefo insulin . In contillation F–G. Inteon  and transgenic overexpression (Tg) mice, which we compared to wild type (WT) mice at ZT0 (corresponding to the nadir in Lpl expression) and ZT12 (corresponding to the peak in Lpl expression). Knockout (Angptl4 was confirmed by quantitative polymerase chain reaction (qPCR). Diurnal gene expression of Lpl was unaltered in Angptl4 KO mice  [de novo) lipogenesis and FA elongation, likely to replenish intracellular lipid stores and efficient combustion during the dark phase. The relative contribution of the various processes is probably context dependent, as cold exposure adds an extra dimension to the diurnal regulation of BAT activity by, for example, increasing the amplitude and expression of genes involved in de novo lipogenesis during the second half of the dark phase [We aimed to gain further insight into the diurnal regulation of metabolic BAT activity; the main findings of the transcriptomics and lipidomics data and our interpretations are summarized as a hypothetical model in e. ZT12) . At thisrk phase , when nuThe question remains what the main driving force is of the diurnal metabolic activity of BAT. Previous experiments indicated that oscillating BAT activity is modulated by glucocorticoids , but is e.g. WAT [The current study primarily focused on BAT because it shows a strong diurnal rhythm in TG-derived FA-uptake as opposed to e.g. WAT . Howevere.g. WAT . However44.1ad libitum access to a chow diet  and water. All mouse experiments were conducted in accordance with the Institute for Laboratory Animal Research Guide for the Care and Use of Laboratory Animals and were approved by the National Committee for Animal experiments.All mice were housed under standard conditions with a 12h:12h light:dark schedule at 22 °C with 2 inhalation at eight time points over a 24-hour period . iBAT was collected and snap-frozen to assess oscillating gene expression by RNA-sequencing, oscillating lipid levels by lipidomics, and oscillating chromatin binding of PPARγ by ChIP-sequencing (see below). In a second and third experiment, male whole-body Angptl4 KO and Angptl4 Tg mice, respectively, were compared with C57BL/6J mice , which were obtained as described previously [Angptl4 Tg mice were compared to littermates and Angptl4 KO mice to mice from another in-house breeding. Angptl4 KO mice do not fully express the Angptl4 gene, resulting in a non-functional ANGPTL4 protein [Angptl4 Tg mice overexpress Angptl4 under its own promotor [Angptl4 KO , Angptl4 Tg  and their WT controls were killed at the onset of the light phase (corresponding to ZT0) and at the onset of the dark phase (corresponding to ZT12) to assess oscillating organ uptake of TG-derived FA (see below).In a first experiment, non-fasted male C57BL/6J mice  were killed by COeviously ,55. Angp protein ,55, wherpromotor . Angptl44.2−1, 10 s; MP Biomedicals™, Santa Ana, California, USA) in a mixture of 400 μL RA1  with 4 μL β-mercaptoethanol in Lysing Matrix D tubes . RNA was quality-controlled on a 2100 Bioanalyzer .Total RNA of iBAT  was isolated using the nucleospin kit  after homogenization by a FastPrep-24™ 5G bead beating grinder and lysis system (4.0 m sMus Musculus reference genome mm10 (Ensembl build 38.88) using GSNAP (version 2017-09-11) [Sequencing quality was assessed with MultiQC , reads w7-09-11)  and read7-09-11)  and the 7-09-11) . Oscilla7-09-11)  (MetaCyc7-09-11) , and tra7-09-11) .4.32, 0.5 mM DTT, 1 × cOmplete™ Protease Inhibitor ), filtered through a Falcon® 70 μm cell strainer , cross-linked in 10 mL 1% formaldehyde in PBS for 15 min and quenched with 1.5 mL 1 M glycine for 5 min. After washing, pelleted nuclei were further lysed twice by resuspension in 1 mL cold fast IP buffer , 5 mM EDTA, 0.5% v/v NP40, 1% v/v Triton X-100, 1 × cOmplete™ Protease Inhibitor) and passing through a 24G syringe. Chromatin was subsequently sheared  in 1 mL shearing buffer , 1 × cOmplete™ Protease Inhibitor). Sheared chromatin was then centrifuged, and 10% of each sample was stored at −20 °C to be used as input. The chromatin was diluted in 7.2 mL dilution buffer , 0.167 M NaCl, 1 × cOmplete™ Protease Inhibitor) and incubated overnight at 4 °C with 8 μg of PPARγ antibody . Chromatin was cleared by centrifugation, and the top 90% was incubated with 100 μL bovine serum albumin-blocked DynaBeads™  for 6 h at 4 °C. Unbound complexes were removed by washing with fast IP buffer. Beads were collected and washed in Tris–EDTA buffer , and immunoprecipitated chromatin was collected by elution with 100 μL beads elution buffer  for 15 min at room temperature two times. Both the input sample and the immunoprecipitated chromatin were de-cross-linked by addition of 4 μL and 8 μL 5 M NaCl, respectively, followed by overnight incubation at 65 °C. Samples were subsequently treated with RNAse A and Proteinase K, and DNA was purified using MinElute® columns from the PCR Purification Kit  using the manufacturer's protocol.ChIP was performed as previously described . BrieflyMus Musculus reference genome mm10 (Ensembl build 38.88) using BWA-MEM (version 0.7.17) [Sequencing quality was assessed with MultiQC , reads w 0.7.17)  and peak 0.7.17) . Reads p 0.7.17) . Peak an 0.7.17)  with the 0.7.17) . Oscilla 0.7.17)  (MetaCyc4.47-cholesteryl ester CE(16:0) (2 nmol), ceramide Cer(d18:1/12:0) (0.118 nmol), ceramide Cer(d18:1/25:0) (0.130 nmol), cardiolipin CL(14:0)4 (0.1 nmol), diacylglycerol DAG(14:0)2 (0.5 nmol), glucose ceramide GlcCer(d18:1/12:0) (0.126 nmol), lactose ceramide LacCer(d18:1/12:0) (0.129 nmol), lysophosphatidicacid LPA(14:0) (0.1 nmol), lysophosphatidylcholine LPC(14:0) (0.5 nmol), lysophosphatidylethanolamine LPE(14:0) (0.1 nmol), lysophosphatidylglycerol LPG(14:0) (0.02 nmol), phosphatidic acid PA(14:0)2 (0.5 nmol), phosphatidylcholine PC(14:0)2 (2 nmol), phosphatidylethanolamine PE(14:0)2 (0.5 nmol), phosphatidylglycerol PG(14:0)2 (0.1 nmol), phosphatidylinositol PI(8:0)2 (0.5 nmol), phosphatidylserine PS(14:0)2 (5 nmol), sphinganine 1-phosphate S1P(d17:0) (0.124 nmol), sphinganine-1-phosphate S1P(d17:1) (0.125 nmol), ceramide phosphocholines SM(d18:1/12:0) (2.129 nmol), sphingosine SPH(d17:0) (0.125 nmol), sphingosine SPH(d17:1) (0.125 nmol), triacylglycerol TAG(14:0)2 (0.5 nmol). 1.5 mL 1:1 (v/v) methanol:chloroform was added before thorough mixing. Each sample was then centrifuged , supernatant was evaporated under a stream of nitrogen at 60 °C and reconstituted in 150 μL of 1:1 (v/v) methanol:chloroform. Lipids were analyzed using a Thermo Scientific Ultimate 3000 binary HPLC coupled to a Q Exactive Plus Orbitrap mass spectrometer. For normal phase separation, 2 μL of each sample was injected onto a Phenomenex® LUNA silica, 250 × 2 mm, 5 μm 100 Å. Column temperature was held at 25 °C. Mobile phase consisted of (A) 85:15 (v/v) methanol:water containing 0.0125% formic acid and 3.35 mmol/L ammonia and (B) 97:3 (v/v) chloroform:methanol containing 0.0125% formic acid. Using a flow rate of 0.3 mL/min, the LC gradient consisted of: 10% A for 0–1 min, reach 20% A at 4 min, reach 85% A at 12 min, reach 100% A at 12.1 min, 100% A for 12.1–14 min, reach 10% A at 14.1 min, 10% A for 14.1–15 min. For reversed phase separation, 5 μL of each sample was injected onto a Waters HSS T3 column . Column temperature was held at 60 °C. Mobile phase consisted of (A) 4:6 (v/v) methanol:water and B 1:9 (v/v) methanol:isopropanol, both containing 0.1% formic acid and 10 mmol/L ammonia. Using a flow rate of 0.4 mL/min, the LC gradient consisted of: 100% A at 0 min, reach 80% A at 1 min, reach 0% A at 16 min, 0% A for 16–20 min, reach 100% A at 20.1 min, 100% A for 20.1–21 min. MS data were acquired using negative and positive ionization using continuous scanning over the range of m/z 150 to m/z 2000. Data were analyzed using an in-house developed metabolomics pipeline written in the R programming language (http://www.r-project.org/). All reported lipids were normalized to corresponding internal standards according to lipid class. Lipid identification has been based on a combination of accurate mass, (relative) retention times, fragmentation spectra and the injection of relevant standards. Oscillation of lipid species was assessed by JTK cycle [Lipidomics was performed as previously described ,68, withTK cycle   were collected and weighed. Half of the tissues  were dissolved overnight at 56 °C in 0.5 mL Solvable  and diluted in 5 mL Ultima Gold . 3H-activity was measured in a liquid scintillation counter  and expressed as a percentage of injected dose per whole organ.Prior to killing, mice of experiment 2 and 3 were fasted for 4 h and received an intravenous injection of TRL-like particles (80 nm) radiolabeled with glycerol tri[eviously   and repeatedly centrifuged  to remove fat. Protein concentrations were determined using the Pierce™ BCA Protein Assay Kit , according to manufacturer's protocol. LPL abundance was analyzed by Western blot using a goat anti-mouse LPL antibody , for which 20 μg protein was loaded. Rabbit anti-mouse glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody  was used for normalization. Anti-goat (1:5000) and anti-rabbit (1:1000) antibodies were used for LPL and GAPDH, respectively. Relative normalized protein levels were quantified by Image Lab software (Bio-Rad).Frozen iBAT samples (approx. 10 mg) (experiment 2 and 3) were lysed in RIPA buffer ), homogenized by a FastPrep-24™ 5G bead beating grinder and lysis system , and the middle layer containing heparin-bound LPL was isolated and used for in vitro assessment of LPL activity. Briefly, 100 μL tissue extract was added to 200 μL substrate solution , 2.5 μCI/mL glycerol-tri--oleate , 0.1% Triton X-100, 0.1 M Tris.HCl pH 8.6 , 1% free fatty acid-free bovine serum albumin , and 20% human serum. After 60 and 120 min, a mix of heptane: methanol: chloroform (in a ratio of 1 : 1.28: 1.37 v/v) and K2CO3 (0.1 M) was added to stop the reaction, samples were diluted in 2.5 mL Ultima Gold , and 3H-activity was measured in a liquid scintillation counter  to calculate TG hydrolase activity.iBAT samples (approx. 10 mg) (experiment 2 and 3) were manually cut with a razor blade in order to keep cells intact, and dissolved in DMEM  containing bovine serum albumin (0.5%) and heparin (0.0004%) to allow for discrimination between extracellular LPL and intracellular LPL. Samples were incubated for 1 h at 37 °C and centrifuged . Subsequently, 1 μg cDNA was synthesized using Moloney Murine Leukemia Virus Reverse Transcriptase  according to the manufacturer's protocol. qPCR was carried out using SYBR green kit  on a CFX96 PCR machine . Lpl (forward primer (FW): CCCTAAGGACCCCTGAAGAC; reverse primer (RV): GGCCCGATACAACCAGTCTA) and Angptl4  expression was normalized to the geometric mean Ct value of Gapdh , ß-tubulin , ß-actin , and 60S acidic ribosomal protein P0 , and expressed relative to the WT ZT0 group using the 2−ΔΔCT method.Total RNA was isolated from frozen sBAT (approx. 10 mg) (experiment 2 and 3) through lysing in TriPure RNA Isolation Reagent  and homogenization by a FastPrep-24™ 5G bead beating grinder and lysis system  and R . Data are presented as means ± SEM.GSE182045 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE182045) and GSE197261 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE197261). The lipidomics dataset discussed in the current study has been deposited in MetaboLights [MTBLS4082 (https://www.ebi.ac.uk/metabolights/MTBLS4082). The remaining datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.The RNA-sequencing and ChIP-sequencing datasets discussed in the current study have been deposited in NCBI's Gene Expression Omnibus (GEO)  and are boLights  and is aConceptualization, R.V.E., W.I.H.P., M.S., S.KE., P.C.N.R., and S.KO.; Formal Analysis, R.V.E., W.I.H.P., J.B.V.K., and T.D.; Investigation, R.V.E., W.I.H.P., M.S., C.J., R.S., T.C.M.S., A.C.M.P., R.H.P.V., H.M., J.B.V.K., M.V.W., N.H.U., and S.KO.; Writing – Original Draft, R.V.E. and W.I.H.P.; Writing – Review & Editing, M.S., S.KE., P.C.N.R., and S.KO.; Supervision, S.KO.; Funding acquisition, P.C.N.R. and S.KO. Both R.V.E. and W.I.H.P. contributed equally and have the right to list their name first in their CV.Netherlands Cardiovascular Research Initiative: an initiative with support of the 10.13039/100002129Dutch Heart Foundation , Dutch Federation of University Medical Centers, the 10.13039/501100001826Netherlands Organisation for Health Research and Development, and the Royal Netherlands Academy of Sciences. M.S. is supported by the 10.13039/501100009708Novo Nordisk Foundation (NNF18OC0032394). S.KO is supported by the 10.13039/100002129Dutch Heart Foundation (2017T016). C.J. and N.H.U. are supported by the 10.13039/501100001659Deutsche Forschungsgemeinschaft (CRC1064 chromatin dynamics and Trans-Regio TRR333 BATenergy).This work is supported by the


In effect, sql-(azpy)(pdia)-Ni exhibited elastic-like properties induced by adaptive guest binding.Single-crystal X-ray diffraction (SCXRD) studies of the α phases revealed that the structural transformations were enabledby the pendant phenyldiazenyl moiety on the pdia2– ligand. This moiety functioned as a type of hinge to enable parallelslippage of layers and interlayer expansion for the following guests: N,N-dimethylformamide, water, dichloromethane, para-xylene, and ethylbenzene. The slippage angle (interplanardistances) ranged from 54.133° (4.442 Å) in the β phase to 69.497° (5.492 Å) in the ethylbenzene-includedphase. Insight into the accompanying phase transformations was alsogained from variable temperature powder XRD studies. Dynamic watervapor sorption studies revealed a stepped isotherm with little hysteresisthat was reversible for at least 100 cycles. The isotherm step occurredat ca. 50% relative humidity (RH), the optimal RH value for humiditycontrol.Herein, we report the crystal structure and guest bindingpropertiesof a new two-dimensional (2D) square lattice ( Out of these, >2500 sql networks have been reported involving N-donor linker ligands, includingboth single-linker and mixed-linker sql networks.33An archetypal class of PCNs is the familyof coordination networkswith square lattice 2(BF4)2], ELM-11, was reported in 2001.34ELM-1136 and its variant, ELM-12,30 [Cu(bpy)2(OTf)2], OTf = triflate, have been widely studied along with otherbpy linked sql analogues.37 More recently, [Co(bipy)2(NCS)2], sql-1-Co-NCS, set a new performance benchmark for C8 hydrocarbon separationsin terms of both selectivity and uptake.17 The mechanism of switching in such networks can be attributed toclay-like expansion/shrinkage between adjacent layers of sql planes and typically results in distinct phases supported by interlayerinteractions in addition to layer–guest interactions.38 Typically, there is little deformation or flexibility associatedwith the sql network itself.An interesting feature of 41 Thus far, only four structures have been reported based upon H2pdia = (E)-5-(phenyldiazenyl)isophthalicacid, H2pdia -1,2-di(pyridin-4-yl)diazene, azpy , 26 of which are single-linker sql nets (Type I-a),33 whereas19 examples are mixed-linker sql nets with dicarboxylateanions as the second linker (Type II-ab).33 Although sql nets based on two distinct azo-bearinglinkers44 and flexible sql nets featuring azo-functionality45 havebeen studied, to the best of our knowledge, the flexibility of thediazene moiety has not been exploited to induce switching behavior.Ligands with diazene(azo) moieties are known to enable deformationand so offer the potential for additional flexibility., H2pdia 1a, but tne, azpy 1a linkersql network sustained by mixed-linkerligands that both contain azo moieties, sql-(azpy)(pdia)-Ni ([Ni(pdia)(azpy)(H2O)], 2– linker ligand upon the nature of the observed structural changes.In this study, we report what is to our knowledge,the first exampleof a switching 3)2·6H2O , (E)-1,2-di(pyridin-4-yl)diazene(azpy) , (E)-5-(phenyldiazenyl)isophthalicacid (H2pdia) , N,N-dimethylformamide (DMF) (5.0 mL), and water (H2O) (5.0 mL) was added to a 20 mL glass vial. The vial was cappedtightly and placed in an oven at 105 °C for 24 h, which was thencooled to room temperature. After rinsing several times with freshDMF, brown single crystals were obtained. Yield: 78%. IR: νmax (cm–1) = 3392, 3292, 2934, 2868, 1650,1596, 1524, 1388, 1219, 1096, 776, 718.A mixture of Ni(NOsql-(azpy)(pdia)-Ni-αDMF) wasexchanged with fresh methanol (MeOH) using a Soxhlet extractor for2 days and then heated to 100 °C under vacuum for 10 h to yield sql-(azpy)(pdia)-Ni-β. IR: νmax (cm–1) = 3363, 1593, 1557, 1531, 1353, 1222, 1044, 1019,918, 832, 761, 715.The as-synthesized open framework (sql-(azpy)(pdia)-Ni-β) was soaked in water for 1 day to yield sql-(azpy)(pdia)-Ni-α2OH. IR: νmax (cm–1) = 3326, 1596, 1553, 1530, 1356, 1222, 1144,1099, 1050, 1015, 918, 778, 721, 684.The activated closed framework (sql-(azpy)(pdia)-Ni-β) was soaked in dichloromethane (DCM) for 1 day to yield sql-(azpy)(pdia)-Ni-αDCM.The activated closed framework (sql-(azpy)(pdia)-Ni-β) was soaked in para-xylene (PX) for 1 day to yield sql-(azpy)(pdia)-Ni-αPX.The activated closed framework (sql-(azpy)(pdia)-Ni-β) was soaked in ethylbenzene (EB) for 1 day to yield sql-(azpy)(pdia)-Ni-αEB.The activated closed framework  system, which gravimetrically measures the uptake andloss of vapor using air as a carrier gas. Pure water was used as theadsorbate for these measurements, and temperature was maintained at298 K by enclosing the system in a temperature-controlled incubator.The mass of the sample was determined by comparison to an empty referencepan and recorded by a high-resolution microbalance with a precisionof 0.01 μg. Sorption isotherms were measured from 0 to 95% relativehumidity (RH) stepwise with a convergence equilibrium criterion dsql-(azpy)(pdia)-Ni were obtainedby solvothermal reaction of H2pdia and azpy in DMF andwater at 105 °C, yielding sql-(azpy)(pdia)-Ni-αDMF. Single-crystal X-ray diffraction (SCXRD)was used to determine the crystal structure of sql-(azpy)(pdia)-Ni-αDMF, which had crystallized in the monoclinicspace group Pc with a = 13.2072(4)4Å, b = 10.1554(3) Å, c = 11.2001(4)4 Å, α = γ = 90°, β = 113.920(10)°,and V = 1373.19(8) Å3 (Table S2). The octahedral mononuclear molecularbuilding block (MBB)46 is comprised ofa Ni2+ cation coordinated to two N-donor atoms (N1 andN4) from two azpy ligands, three carboxylate O-donor atoms  from two pdia2– ligands, and one O-donoratom (O5) from an aqua ligand. The formula is [Ni(pdia)(azpy)(H2O)]·DMF . Hydrogenbonds were observed between each coordinated aqua molecule and O-atomsof coordinated pdia2– ligands, both within individual sql layers (O···O = 2.614(5) Å) and betweenneighboring sql layers (O···O = 2.783(6)Å, Table S3). A 3D network with primitivecubic, pcu, topology structure results. The perpendiculardistance between adjacent sql planes formed by Ni cationsis 5.120(6) Å, and the dihedral angle between the sql plane and the plane of the parallelogram formed from two pairs ofNi2+ cations from adjacent sql layers is 66.075(13)°(Table S3). sql-(azpy)(pdia)-Ni-αDMF has an effective pore size of ca. 6 ×7 Å2 along the c-axis, and the calculatedguest-accessible void volume is 19.5% .The bulk experimental powder X-ray diffraction (PXRD) pattern of αDMF is consistent with thatcalculated from SCXRD data ]·DMF S3. Hydrosql-(azpy)(pdia)-Ni-αDMF wassoaked in methanol, exchanged twicedaily for 2 days, and then activated at 100 °C under vacuum.The activated phase had transformed to a nonporous phase, sql-(azpy)(pdia)-Ni-β, as determined by SCXRD. sql-(azpy)(pdia)-Ni-β is a contorted version of sql-(azpy)(pdia)-Ni-αDMF with the same connectivity and spacegroup but different unit-cell parameters and a 14.6% reduction inunit-cell volume when compared to αDMF (Table S2). The αDMF to β transformationwas accompanied by a folding motion of the channels along the c-axis, resulting in reduction of guest-accessible volumefrom 19.5% (αDMF) to 0%(β) as calculated by Mercury software . The transformation from αDMF and β can be attributedto a deformation in the pdia2– and azpy ligandswhereby “hinge-like” rotation occurred along the azobond. In effect, the azo bonds in pdia2– and azpyact as axles . The dihedralangles of the isophthalate ring and phenyl ring of pdia2– ligand in αDMF and β changed substantially from 8.161 to 48.930° (47.373°)(Table S4). The azo bond, which connectsthe two pyridine rings in azpy ligand, also changes orientation duringthe transformation from αDMF to β, the dihedral angles between these two pyridinerings being 4.756 and 69.133°, for αDMF and β, respectively (Table S4).Prior to conducting gas sorption experiments, software S4. The t2– in αDMF and β are superposed and compared, themaximum torsional angle about theazo bond is ca. 33° –2.832(7) Å), but the distancebetween adjacent sql planes in αDMF and β decreased from 5.120(6)to 4.442(10) Å  to 54.133(21)° (Table S3). In effect, slippage between sql layers had occurred during the transformation from αDMF to β.When the isophthalate ringsof pdia ca. 33° 2c. Upon  ca. 49° 2d. These– ligand 2a,b. TheXRD data 2f. The i42(10) Å 3c,a. Thesql-(azpy)(pdia)-Ni, crystals of sql-(azpy)(pdia)-Ni-β were soakedin the following solvents: H2O, DCM, PX, and EB. SCXRDexperiments revealed that four additional open phases were obtained,namely, sql-(azpy)(pdia)-Ni-α2OH, sql-(azpy)(pdia)-Ni-αDCM, sql-(azpy)(pdia)-Ni-αPX, and sql-(azpy)(pdia)-Ni-αEB. SCXRD data revealed that folding/unfoldingof the pores and slippage between layers had occurred in such a mannerthat sql-(azpy)(pdia)-Ni, in effect, adapts its structurefor each guest. Cell volumes were as follows: β = 1199.07(12) Å3; α2OH = 1296.43(5) Å3; αDCM = 1350.30(7) Å3; αDMF = 1373.19(8)Å3; αPX = 1389.00(9) Å3; and αEB = 1469.2(3) Å3 (Table S2). These cell volumes correspond to the relative molecularvolume of each guest: H2O (2 × 18.4 Å3); DCM (1 × 58 Å3); DMF (1 × 72.3 Å3); PX (1 × 110.2 Å3); and EB (1 ×110.2 Å3), as calculated by XSeed,47 except for H2O, which was calculated from theCSD.49 Although PX and EB exhibit the same molecularvolume, the shape of PX enabled a better fit than EB, which in turnrequired a larger pore volume. Short contact distances between guestmolecules and pore walls were observed for αDMF (d(O···H) = 2.516 Å), α2OH (d(O···O) = 2.969Å), αEB (d(C–H···π) = 3.005 Å), αDCM (d(O···H) = 2.294 Å), and αPX (d(O···H) = 2.927 Å) (Table S3). Disorderof ligands was observed in αDMF, β, α2OH, and αEB related to host–guest interactions and framework flexibility.Overall, the analysis of the crystal structures of the six phasesindicates that adaptive binding (S5) and slippage motion are enabled by the pendant phenyldiazenyl moietyof the pdia2– ligand.To further explore the mechanism of flexibilityin sql-(azpy)(pdia)-Ni canbe quantified by the sql layer separation and dihedralangle formed by the sql plane and the plane formed bypairs of Ni2+ cations from adjacent sql layers(Table S3). At one extreme, αEB possesses the biggest void volume (21.8%),longest interlayer distance (5.492(15) Å), and most obtuse dihedralangle (69.497(20)°) (β is nonporous(0%) and has the shortest interlayer distance (4.442(10) Å) andthe most acute dihedral angle (54.133(26)°) (Table S3), whereas the other structuralparameters are intermediate between the extremes. The motion between sql layers can be described as being analogous to parallelmotion linkage, a concept from engineering and architecture (50The continuous natureof the slippage/expansion in 97(20)°) 3f. At th33(26)°) 3a. The iitecture 3h.50sql networkslike ELM-11,36ELM-12,51 and sql-1-Co-NCS,36n-butane,52 and acetylene (C2H2).53 With increasing pressure of CO2,the phase change from closed to open phases resulted in distancesbetween sql layers of 4.427, 5.676, 5.685, and 6.960Å. In ELM-12, vacuum heating prompted interlayer sql distances to decrease from 7.2 to 6.7 Å and thento 5.9 Å, corresponding to three distinct phases. C8 aromaticswere reported to induce sql-1-Co-NCS to exhibit fourdistinct phases with interlayer sql distances of 4.46(closed) to 9.15 (PX), 9.21 , 9.26, and 6.25 (EB) Å. In contrast,anchored by an interlayer H-bond, layer expansion was not as substantialin sql-(azpy)(pdia)-Ni. Rather, slippage of sql layers was induced by a series of guests to increase molecular volume. Further, PXRD experimentsconducted after immersing sql-(azpy)(pdia)-Ni-β in aqueous solutions with a range of pH values revealed that sql-(azpy)(pdia)-Ni retained its crystallinity after exposureto pH values ranging from 2 to 11 . Crystallinity was also retained under accelerated humidity stabilitytesting conditions over 48 h . The thermal stability of sql-(azpy)(pdia)-Ni was evaluated by thermogravimetric analysis (TGA) and variable temperaturePXRD (VT-PXRD). TGA results revealed that sql-(azpy)(pdia)-Ni-αDMF exhibited a mass loss of 11.4% at 115°C, corresponding to one DMF molecule per formula unit . TGA conducted on sql-(azpy)(pdia)-Ni-β showedno mass loss below thermal decomposition at 513 K . Attempts to analyze α2OH by TGA showed identical resultsto β, which indicated desorption of water at ambientconditions . VT-PXRD conducted on sql-(azpy)(pdia)-Ni-α2OH revealed that a transformationto β occurred at 298 K at the onset of nitrogen(N2) flow without application of heat, following which β was found to be stable up to 473 K .The bulk phasepurities of sql-(azpy)(pdia)-Ni, we studied its gas sorptionproperties. sql-(azpy)(pdia)-Ni-β revealed negligibleuptake for N2 at 77 K, whereas a stepped isotherm was observedfor CO2 at 195 K, with the inflection occurring at verylow pressure , confirming that sql-(azpy)(pdia)-Ni acts as a stimulus-responsivesorbent. In addition, the CO2 sorption at 273 K exhibitedthe onset of a step at 681 mmHg, which is consistent with a structuraltransformation . This stepwas not observed below 1 bar at 298 K. The single point pore volumecalculated at saturation (P/P0 = 0.9) from the 195 K CO2 isotherm is 0.118 cm3 g–1, which agrees well with the crystallographicallydetermined accessible void volume in sql-(azpy)(pdia)-Ni-α2OH (0.124 cm3/g),indicating that the CO2-included phase resembles α2OH.Encouraged by the stability and responsivesolution-phase uptakebehavior of sql-(azpy)(pdia)-Ni isof topical interest since water sorbents are being studied fortheir potential utility in water harvesting and dehumidification.55 To evaluate the sorption characteristics of sql-(azpy)(pdia)-Ni, dynamic water vapor sorption experiments were conducted on sql-(azpy)(pdia)-Ni-β. A stepped isotherm with an abruptuptake was observed at 298 K. The step occurred between 50 and 55%RH with an uptake of 2 wt % at 50% RH and an uptake of 9 wt % (162 cc/cc) at 55% RH. The uptakedifference of 7 wt % (126 cc/cc) is consistent with two water moleculesper formula unit (pdia)-Ni-α2OH, which exhibits both water–waterand water–framework short contacts , suggesting a wide operating temperature rangefor indoor humidity control.In contrast to the pore filling (Type V) mechanism,which is oftenobserved in rigid sorbent frameworks, such a stepped isotherm, alongwith the aforementioned structural changes, indicates that a nonporous-to-porousswitching event occurs from . 45% RH 4a. This sql-(azpy)(pdia)-Ni-β were studied by subjecting a sampleto humidity swing conditions,wherein RH was varied between 0 to 90%. As shown in sql-(azpy)(pdia)-Ni can not only rapidly capture water vapor from the gas phase (asmuch as 9.4 wt % in 10 min or 169.2 cc/cc at 90% RH and 298 K) butalso can be fully regenerated in less than 10 min once the RH is decreasedto 0%. To evaluate the reusability of sql-(azpy)(pdia)-Ni for water sorption, humidity swing conditions of 298 K, 0–90%RH were applied to the sample for 100 cycles of 20 min each . No loss of working capacity was observedover the course of the experiment despite undergoing structural transformationsduring each cycle . To approximate real-world dehumidificationconditions, sorption kinetics from 40 to 60% were also studied. Theuptake was found to reach 9 wt % (162 cc/cc) within 60 min at 60%RH, and desorption to a loading of 1 wt % (18 cc/cc) occurred within25 min at 40% RH. Working capacity was not significantly reduced over12 cycles under these conditions .The sorption kinetics of ch cycle 4c. The rsql-(azpy)(pdia)-Ni was further studied by monitoring itsstructure by PXRD during repeatedexposure to dry and humid conditions. First, sql-(azpy)(pdia)-Ni was exposed to dry N2 flow at room temperature (10 min),exhibiting a PXRD pattern matching with the β phase.Then, instead of N2 flow, a PXRD pattern of sql-(azpy)(pdia)-Ni was measured in a humid atmosphere at the same temperature. α2OH was obtained,as expected. PXRD patterns corresponded to the β and α2OH phaseswithout loss of crystallinity over 4 cycles of the experiment (sql-(azpy)(pdia)-Ni-β and exposed to successive hydration and evacuation showed correspondingchanges from β to α2OH and back to β didnot exhibit signs of fragmentation despite somewhat diminished crystalquality (Table S2). Time-lapse spectraFourier Transform Infrared (FTIR) studies support the observed fastkinetics of water loading and unloading, showing a progressive reductionof the strong O–H water stretching peak at 3326 cm–1 until its disappearance within 10 min under ambient conditions , leaving onlypeaks due to coordinated water in β (3356 cm–1). These data reveal that regeneration of sql-(azpy)(pdia)-Ni can be easily realized by simply adjusting RH. Favorable intrinsicheat management arising from the endothermic structural transformationfrom β to α2OH is anticipated. To date, over 370 desiccantMOMs have been investigated, but fewer than 10% exhibit water-inducedflexibility (Table S5), and, to our knowledge,no two-dimensional (2D) FMOMs have previously been studied for humiditycontrol. sql-(azpy)(pdia)-Ni is therefore a potentialcandidate for indoor humidity control.The mechanism of water sorption by the switching of periment 5b. In adsql-(azpy)(pdia)-Ni was synthesized using the azpy and pendant-bearingpdia2– ligands. Adaptive binding of guest moleculesresulted in six distinctphases of sql-(azpy)(pdia)-Ni corresponding to phasesloaded with DMF, H2O, DCM, PX, EB, and a nonporous phase,as elucidated by SCXRD and PXRD. The continuum of phases exhibitedby sql-(azpy)(pdia)-Ni was facilitated by the pendantphenyldiazenyl moiety on the pdia2– ligand and interlayerhydrogen bonds between adjacent sql layers. This elastic-likemotion is key to regulating access to the pores in each phase andwas also observed during gas and H2O sorption. In particular,the stepped water sorption isotherm with inflections in the range40–60% RH makes sql-(azpy)(pdia)-Ni a potentialcandidate for indoor humidity control applications. Studies of stabilityand kinetics on sql-(azpy)(pdia)-Ni show excellent recyclabilityand retention of crystallinity. We attribute the elastic-like propertiesof sql-(azpy)(pdia)-Ni to the characteristic of rotationin diazo moieties and the pendant nature of one of the linker ligands,which enables a hinge-like mechanism of structural transformation.The amenability of sql nets to crystal engineering approacheswill likely afford more members of this family of switching sorbentmaterials.In conclusion, a new switching 2D MOF


Spinal cord injury (SCI) elicits a robust neuroinflammatory reaction which, in turn, exacerbates the initial mechanical damage. Pivotal players orchestrating this response are macrophages (Mφs) and microglia. After SCI, the inflammatory environment is dominated by pro-inflammatory Mφs/microglia, which contribute to secondary cell death and prevent regeneration. Therefore, reprogramming Mφ/microglia towards a more anti-inflammatory and potentially neuroprotective phenotype has gained substantial therapeutic interest in recent years. Interleukin-13 (IL-13) is a potent inducer of such an anti-inflammatory phenotype. In this study, we used genetically modified Mφs as carriers to continuously secrete IL-13 (IL-13 Mφs) at the lesion site.Mφs were genetically modified to secrete IL-13 (IL-13 Mφs) and were phenotypically characterized using qPCR, western blot, and ELISA. To analyze the therapeutic potential, the IL-13 Mφs were intraspinally injected at the perilesional area after hemisection SCI in female mice. Functional recovery and histopathological improvements were evaluated using the Basso Mouse Scale score and immunohistochemistry. Neuroprotective effects of IL-13 were investigated using different cell viability assays in murine and human neuroblastoma cell lines, human neurospheroids, as well as murine organotypic brain slice cultures.In contrast to Mφs prestimulated with recombinant IL-13, perilesional transplantation of IL-13 Mφs promoted functional recovery following SCI in mice. This improvement was accompanied by reduced lesion size and demyelinated area. The local anti-inflammatory shift induced by IL-13 Mφs resulted in reduced neuronal death and fewer contacts between dystrophic axons and Mφs/microglia, suggesting suppression of axonal dieback. Using IL-4Rα-deficient mice, we show that IL-13 signaling is required for these beneficial effects. Whereas direct neuroprotective effects of IL-13 on murine and human neuroblastoma cell lines or human neurospheroid cultures were absent, IL-13 rescued murine organotypic brain slices from cell death, probably by indirectly modulating the Mφ/microglia responses.Collectively, our data suggest that the IL-13-induced anti-inflammatory Mφ/microglia phenotype can preserve neuronal tissue and ameliorate axonal dieback, thereby promoting recovery after SCI.The online version contains supplementary material available at 10.1186/s12974-022-02458-2. Following spinal cord injury (SCI), a wide variety of secondary injury processes are provoked, which significantly worsens patients’ outcomes . Among tIn particular, M2a Mɸs have been identified as essential players in neuroregeneration by counteracting inflammation and promoting wound healing , 7. This+ Mφs/microglia. Thus, it was tempting to speculate that accumulated M2a Mφs were critical for functional repair [In this study, the goal was to modulate perilesional Mφs/microglia and specifically exploit their M2a phenotype to influence motor recovery beneficially. For this aim, we used the canonical T-helper cell type 2 cytokine interleukin-13 (IL-13), which is one of the main inducers of this M2a subset . Followil repair . Therefol repair .We show for the first time that the use of genetically modified Mφs as carriers of IL-13 is a potent strategy to improve histopathological and functional recovery after SCI. Using IL-4Rα-deficient mice, we demonstrate that IL-13 signaling is essential to reduce neuronal death and suppress axonal dieback by promoting an anti-inflammatory phenotypic shift of perilesional Mφs/microglia. Overall, these findings underline the therapeutic potential of IL-13 and Mφ/microglia modulation in regeneration after CNS trauma.+/GFP mice were kindly provided by prof. Brône (Uhasselt). In vivo experiments were performed with either female 10–14-week old WT C57BL/6j mice (Janvier Labs) or IL-4Rα control and knockout (KO) BALB/cJ mice  [In vitro experiments were done using female 8–12-week old wild type (WT) BALB/c (Envigo) or C57BL/6J mice (Janvier Labs). CX3CR1oratory) . As male2. Every 2–3 days, partial or complete medium change was done. After 7 days, naive M0 Mɸs were obtained. To induce M1 or M2 polarization, Mɸs were stimulated with 200 ng/ml lipopolysaccharide  for 24 h or 33.3 ng/ml recombinant IL-13  for 48 h, respectively. Conditioned medium (CM) was collected after stimulation.Bone-marrow-derived Mɸs were obtained from female mice. In brief, primary bone marrow cells were isolated by flushing the femur and tibia with 1× phosphate-buffered saline . Cells were cultured in RPMI 1640 medium (Lonza) supplemented with 10% heat-inactivated fetal calf serum , 1% penicillin/streptomycin , and 15% L929 conditioned medium at 37 °C and 5% CO5 cells/well in standard medium. Positive selection of the transduced cells was made using puromycin  for 72 h. CM was collected after puromycin selection.To generate IL-13 overexpressing and Arg1 overexpressing Mɸs, naive Mɸs were transduced with pIRES-mIL-13-puro or pIRES-Arg1-puro lentiviral particles provided by the Laboratory for Viral Vector Technology & Gene Therapy  according to previously optimized protocol with minor adaptations . Mɸs werCells were lysed using QIAzol (Qiagen), and RNA was extracted using the RNeasy mini kit (Qiagen), according to the manufacturer’s instructions. Conversion to cDNA was done with qScript cDNA supermix (Quanta Biosciences). Quantitative PCR was performed on a StepOnePlus detection system (Applied Biosciences) under universal cycle conditions. Primer sequences can be found in the Additional information . The protein concentration was determined by the Pierce™ BCA assay kit (ThermoFisher Scientific), according to the manufacturer's guidelines. Samples (4–10 µg) were separated on 7.5% or 12% (dependent on size target) sodium dodecyl sulfate–polyacrylamide gels by electrophoresis (1 h at 200 V) and transferred to a polyvinylidene fluoride membrane . After blocking using 5% non-fat dry milk in 1× tris-buffered saline supplemented with 0.05% Tween 20 (TBS-T), blots were incubated with primary antibodies overnight at 4 °C. Used antibodies included: mouse anti-Arg1 , mouse anti-inducible nitric oxide synthase , and mouse anti-β-actin . After washing, secondary antibodies  were applied for 1 h at RT (room temperature). β-Actin was used as a loading control. Visualization was done using the Pierce™ ECL plus Western blotting substrate (ThermoFisher Scientific). Images were taken with an Amersham Imager 680  and analyzed using the ImageQuant TL software. Pictures displayed in the figures were digitally enhanced to improve visibility.IL-4 and IL-13 secretion in the supernatants of cell cultures was measured using ELISA kits (ThermoFisher Scientific), according to the manufacturer’s instructions.Nitrite secretion in the supernatants of cell cultures was measured using the Griess reagent system kit (Promega), according to the manufacturer’s instruction.A T-cut spinal cord hemisection injury was performed as previously described , 18. Bri+) Mφs. As previously described, a motorized stereotaxic injector pump (Stoelting) with a 10 µl Hamilton syringe (34-gauge needle) was positioned 1–3 mm rostral to the lesion [4 in 2.5 µl) were injected over a 4 min period, after which the needle was kept in place for again 4 min to allow pressure equilibration and prevent backflow. Investigators were blinded to the treatment groups during the complete experiment.For in vivo transplantation, Mφs were isolated from the same mouse strain as the recipient mice in the in vivo experiment, being either C57BL/6J or BALB/c. Mφs were cultured using Nunc™ Dishes with UpCell™ Surface (ThermoFisher Scientific) to avoid enzymatic harvesting. Immediately after SCI induction, mice were randomized and received an intraspinal injection of vehicle or  for locomotion  . Scores At 8 or 28 dpi, mice received an overdose of dolethal (Vetiquinol B.V.) and were transcardially perfused using ringer solution supplemented with heparin, followed by perfusion with 4% paraformaldehyde (PFA) in PBS. Longitudinal cryosections of 10 µm were obtained. Immunofluorescent stainings were done as previously described , 18.+ cells, sections were only counterstained with DAPI  for 10 min. To investigate lesion size, demyelinated area, astrogliosis, and immune cell infiltration, sections were blocked using 10% protein block (Dako) in PBS containing 0.5% Triton-X-100 for 1 h at RT. Primary antibodies included mouse anti-glial fibrillary acidic protein , rat anti-myelin basic protein , rabbit anti-Iba-1 , goat anti-ionized calcium binding adaptor molecule 1 , rabbit anti-neurofilament , mouse anti-NeuN , rabbit anti-cleaved caspase 3  were diluted in PBS with 1% protein block and 0.5% Triton-X-100 and were incubated overnight at 4 °C. To investigate Arg1+ and major histocompatibility complex class ll (MHCII)+ cells, an extra permeabilization step using 0.1% Triton-X-100 in TBS for 30 min was performed before blocking the sections for 1 h with 10% protein block in TBS. Primary antibodies, goat anti-Arg1  and rat anti-MHCII , were dissolved in TBS containing 10% non-fat dry milk powder and were incubated overnight at 4 °C. Following washing, secondary antibody incubation was done for 1 h at RT. The following secondary antibodies were used: goat anti-rat Alexa fluor 488 , goat anti-mouse Alexa fluor 568 , goat anti-mouse GaM IgG1 fluor 488 , donkey anti-rabbit IgG 555 , goat anti-rabbit Alexa fluor 488 , donkey anti-goat Alexa fluor 488  and donkey anti-goat Alexa fluor 555 . The specificity of secondary antibodies was tested by omitting the primary antibody. Counterstaining with DAPI was performed for 10 min. Pictures were taken using a Nikon Eclipse 80i fluorescence microscope and analyzed with NIS-Elements Viewer 4.0 software or using a LEICA DM4000 B LED microscope and LAS X software (Leica Microsystems).To determine the presence of GFPTo investigate lipid load, an oil red O (ORO) staining was performed. Sections were incubated with a 0.33% ORO solution (Sigma-Aldrich) for 15 min, followed by a 5 min incubation with 60% isopropanol. To wash away any unbound particles, the sections were rinsed with distilled water. Pictures were taken using the Leica DM 2000 LED microscope (Leica Microsystems).+ cells at the injection, perilesional, and lesion site were counted. To quantify lesion size (GFAP− area) and demyelinated area (MBP− area), 5–7 sections per animal containing lesion center and consecutive rostral and caudal area were analyzed. The proximal and distal end of the lesion was detected by identifying the area, where the integrity of the dorsal side of the spinal cord tissue is disrupted. As no intensity was measured, contrast was altered differently among groups. To determine astrogliosis (GFAP expression) and Mφ/microglia presence (Iba-1 expression or Iba-1+ cells), an intensity analysis was performed, or the number of positive cells was counted using ImageJ [+ or MHCII+ cells were counted caudally and rostrally from the lesion site. Quantification of foam cell presence was carried out using ImageJ software according to the method previously described by Deutsch et al. with slight adaptations [+NeuN+ cells was counted at both rostral and caudal areas from the lesion epicenter and normalized to the amount of NeuN+ cells. To quantify the interaction between axons and Mφ/microglia, the number of contacts between NF+ dystrophic axons and Iba-1+ cells was counted in the perilesional environment. Of note, dystrophic axons were identified by their globular and bulbus morphology extending from an axon fiber [+ cell contained a DAPI nucleus. For analysis, two standardized areas, rostral and caudal, from the lesion epicenter were used. The mean number of contacts in these two areas per animal was quantified.Quantification was carried out on the original, unedited pictures. Pictures displayed in the figures were digitally enhanced to improve visibility. Quantification of histopathological parameters was performed as previously described and was done by investigators blinded to experimental groups , 18. To g ImageJ . To idenptations . To deteon fiber , 23. A c2. For immunocytochemistry, cells were seeded in a 24-well plate at 3 × 104 cells/well for 48 h. To perform cell viability assay, cells were seeded in a 96-well plate at 5 × 103 cells/well and left untreated for 24 h to attach.The murine Neuro2A cell line  was cultured in RPMI 1640 medium supplemented with 10% FCS and 1% penicillin/streptomycin. The human SH-SY5Y cell line (Sigma-Aldrich) was cultured in DMEM (Gibco) supplemented with 10% FCS and 1% penicillin/streptomycin. Cells were maintained at 37 °C and 5% COCells were fixed with 4% PFA and blocked for 1 h at RT with 10% protein block in PBS. Anti-IL-13Rα1 and anti-IL-4Rα  antibodies were diluted in PBS with 1% protein block and were incubated overnight at 4 °C. Following washing, secondary antibody  incubation was done for 1 h at RT. The specificity of the secondary antibody was tested by omitting the primary antibody. Counterstaining with DAPI was performed for 10 min. Pictures were taken using a LEICA DM4000 B LED microscope and LAS X software (Leica Microsystems).l-glutamine, and distilled water, Gibco) and using a tissue chopper , 350 μm thick slices were made. These were cultured on inserts  in incubation medium containing 50% MEM, 25% HBSS, 25% heat-inactivated horse serum, 4 mM l-glutamine, 4 ng/ml insulin, 800 ng/ml ascorbic acid, 2.4 mg/ml glucose and 1% penicillin/streptomycin, Gibco) [Hippocampal entorhinal cortex slices were prepared from 8 days old pups. In short, brains were isolated in dissection medium  . In addi4 cells/well), stably expressing luciferase in an ultra-low attachment 96-well plate. Half medium changes with cNEM  were performed every other day. Cultures were kept under constant orbital shaking (88 rpm).Neurospheroids were generated by seeding human-induced pluripotent stem cell-derived neural stem cells  was diluted in 10% milk in TBS and was applied overnight at 4 °C. Following washing, secondary antibody  incubation was done for 1 h at RT. Counterstaining with DAPI was performed for 10 min. Pictures were taken using a BX51 fluorescence microscope equipped with an Olympus DP71 digital camera (Olympus).S-nitroso-N-acetylpenicillamine, 100–800 μM, Abcam). Cells were either pre-treated for 24 h or co-treated with various rIL-13 concentrations . After 48 h or 72 h, 0.5 mg/ml MTT  was added to the standard medium for 4 h, after which the cells were lysed and the formazan crystals dissolved in a mixture of DMSO and glycine (0.1 M). Absorbance was measured at a wavelength of 570 nm.In the neuroblastoma monoculture, cell death was induced via incubation with different SNAP concentrations  for 4 h. After washing, PI  was added for 10 min. Pictures were taken using a Nikon Eclipse 80i fluorescence microscope. The PI uptake (fluorescence intensity) from the cells of the cornu ammonis region (CA1–CA3) of the hippocampus was analyzed using ImageJ software.In the brain slices culture, cell death was induced via incubation with 100 μM NMDA  in a humidified Bactron IV anaerobic chamber  containing 0% O2, 5% CO2, 95% N2. After OGD, glucose  was administered, and spheroids were brought back to atmospheric O2 level (21%). After 90 min of reoxygenation, human rIL-13  was added to the medium for 48 h. Luminescence of the neurospheroids was determined 96 h following reoxygenation by incubating with 150 µg/ml Beetle luciferin (Promega) for 48 h.Neurospheroids (4 weeks) were subjected to control or oxygen–glucose deprivation (OGD) conditions for 6 h. OGD conditions were achieved by incubating the neurospheres in glucose-free NSC medium .Data analysis was performed using GraphPad Prism version 7 (GraphPad Software). D’Agostino and Pearson omnibus test was used to assess normality. The BMS results were analyzed using a two-way ANOVA for repeated measurements with a Bonferroni post hoc test. GFAP and Iba-1 intensity were analyzed using a two-way ANOVA with a Bonferroni post hoc test. All other data were analyzed using either a one-way ANOVA with a Bonferroni post hoc test or with a non-parametric Kruskal Wallis test with a Dunn’s multiple comparison test. Data are presented as mean ± standard error of the mean (SEM). Differences with Mφs are reported to migrate towards inflammatory sites after CNS trauma . TherefoNext, an extensive phenotypical analysis of typical anti- and pro-inflammatory markers expressed by the IL-13 Mφs was performed before in vivo transplantation. Gene data analysis showed that expression of Arg1, FIZZ1, and Ym1 was significantly increased in IL-13 Mφs, as well as in M2 Mφs, compared to M0 Mφs  and classically (MHCII+) activated phagocytes at the lesion site was determined  and dystrophic axons (NF+ cells showing end bulbs) at the lesion site [The abovementioned results indicate that IL-13 Mφs transplantation enforced an anti-inflammatory perilesional milieu. Next, we investigated whether this change in environment also provided neuroprotection. First, the neuroprotective effect of IL-13 Mφs was determined through double staining for cleaved caspase 3 and NeuN to detect apoptotic neurons. Mice treated with IL-13 Mφs had reduced neuronal cell death compared to vehicle Fig. a, c, e. ion site . IL-13 M. This can be attributed to either IL-13 secretion or the anti-inflammatory phenotype of the IL-13 Mφs . To investigate this, we used two different approaches. First, IL-13 Mφs were administered to IL-4Rα KO mice. These mice lack not only IL-4 signaling but also IL-13 signaling as IL-13Rα1 activation requires heterodimerization with the IL-4Rα, which is prevented in the IL-4Rα KO model [We have shown that IL-13 Mφs have anti-inflammatory and neuroprotective effects in vivo, accompanied by improved functional recovery after SCIKO model . After SThese results indicate that IL-13 is critical for functional recovery and neuroprotective effects induced by IL-13 Mφs, which are absent in mice deficient for IL-4Rα signaling. Next, we investigated in vitro whether IL-13 was directly responsible for neuroprotection. We used murine and human neuroblastoma cell lines and induced cell death via NO, which plays a vital role in secondary injury neurotoxicity . Of noteThe abovementioned results suggest that IL-13 does not have direct neuroprotective actions. Therefore, we used a murine brain slice model, which is a more complex in vitro trauma model to maintain the interaction between CNS neural and immune cells to include analyses of indirect effects induced by IL-13 on neurons. In this case, we used a model of NMDA-induced toxicity as NMDA contributes to secondary cell death via glutamate excitotoxicity . In thisIt is generally accepted that neuroinflammation is a significant driver of secondary injury-related damage in SCI. During the last years, the potential of IL-13 as a treatment for neuroinflammation has been shown by us and others in different animal models of neurodegeneration , 19, 32.IL-13 secretion by Mφs has not been previously reported under physiological conditions, only in the context of pulmonary fibrosis . Through+-M2 Mφs, only 20–40% maintained their polarization status [As the inflammatory imbalance following SCI impedes regenerative processes, increasing the fraction of the anti-inflammatory Mφ/microglia phenotype has become a major therapeutic strategy. Ma et al. demonstrated that intravenous injection of M2 Mφs improved functional recovery after SCI . Howevern status . This isn status . Importan status –44. For n status , 43. Surn status , 45. Forn status . Still, + cells) and a decreased pro-inflammatory cell number (MHCII+ cells) without affecting the global Mφs/microglia cell presence. Notably, the increase in Arg1+ cells was absent in the mice who received M2 Mφs. This immunomodulation of local Mφs/microglia by IL-13 is in line with previous stroke and TBI reports [The M1:M2 ratio is a crucial determinant of functional recovery after SCI [ reports –14. Cons reports .+ cells, it may be expected that foam cell formation is reduced. Indeed, the ORO staining results showed an overall reduction in lipid load at the lesion site following IL-13 Mφ transplantation. Even though the underlying mechanism remains to be resolved, several studies indicate a role of IL-13 in phagocytosis. The importance of IL-13 in stimulating efferocytosis was described in a TBI animal model where repeated intranasal rIL-13 treatment increased the NeuN+Iba-1+ area [Lesional lipid load was analyzed as an indirect measurement of foamy cells, which are detrimental in SCI . Mφs pha-1+ area . Mechani-1+ area . In addi-1+ area . Based o+ neurons in IL-13 Mφ-treated mice. Furthermore, damaged axons retract from the injury site after SCI, a process called axonal dieback [+ cells compared to vehicle. However, it reduced the number of contacts between dystrophic axons and phagocytes. Since the number of Iba-1+ cells was not influenced, this suggests that axonal dieback, a limiting factor for regeneration, was reduced [Notably, the beneficial immunomodulatory effects of IL-13 Mφs were accompanied by neuroprotection. In animal models of TBI and stroke, IL-13 treatment also facilitated anti-inflammatory effects and reduced neuronal cell loss , 13. Lik dieback , 51. The dieback . In addi dieback . IL-13 M reduced . Therefo reduced .α KO mouse model. IL-13 binding to its receptor, IL-13Rα1, will evoke the dimerization with the IL-4Rα for downstream pathway activation [α KO mice did not show differences in functional recovery after SCI. This is in line with Fenn et al., who did not observe reduced recovery after SCI in adult IL-4Rα KO mice [α KO mice, highlighting the role of proper IL-4Rα signaling. Besides their IL-13 secretion, IL-13 Mφs represent anti-inflammatory Mφs which overexpress Arg1 compared to rIL-13-stimulated M2 Mφs. To investigate whether this polarization was involved in their beneficial effects, Arg1 overexpressing Mφs, which do not secrete IL-13, were also grafted following SCI. However, no significant impact on functional recovery was observed. These results point towards IL-13 signaling as the critical mechanism by which IL-13 Mφs induce immunomodulation, neuroprotection, and functional recovery after SCI. Consistently, IL-13 delivery by MSCs was able to modulate Mφ/microglia polarization during the active inflammation phase in a cuprizone model [To analyze whether the beneficial effects of IL-13 Mφs were either dependent on IL-13 itself or their altered M2a phenotype, we used an IL-4Rtivation . Deletiotivation . Howevertivation . In addi KO mice . The funne model . As we wFinally, our results indicate that the neuroprotective effect by IL-13 Mφs was not mediated by direct action of IL-13 on neurons. In both murine and human neuroblastoma cell lines, neither pre- nor co-treatment with IL-13 protected against NO-induced cell death. Similarly, no protective effect of IL-13 on the cell viability of OGD-subjected neurospheroids was observed. However, in an murine organotypic brain slice model, IL-13 (5 ng/ml) rescued neurons from NMDA-induced cell death. This suggests that IL-13 indirectly affects the survival of neurons via IL-13R-expressing microglia, which are present in organotypic brain slice culture but not in the neuronal monocultures. Consistent with these findings, Shin et al. reported that the induction of IL-13 in microglia contributed to the survival of neurons upon inflammation . AccordiTo summarize, this study shows for the first time that perilesional transplantation of genetically modified Mφs that secrete IL-13 is a promising approach to improve functional recovery after SCI. Our data suggest that IL-13 elicits indirectly neuroprotection via stimulating anti-inflammatory responses in Mφs/microglia. In addition, IL-13 also possibly ameliorated axonal dieback facilitating regeneration. Our data strongly support the therapeutic use of IL-13 and consider it a prime candidate for effective immunomodulation after SCI to improve repair.Additional file 1: Table S1. Primers used for qPCR.Additional file 2: Figure S1. IL-13 Mφs have an anti-inflammatory phenotype. a–f Mφs were isolated from C57BL/6J mice. qPCR showed that gene expression of the anti-inflammatory markers FIZZ1 (a) and Ym1 (b) were significantly increased in the IL-13 Mφs compared to M0 Mφs, whereas CD206 (c) was not. Pro-inflammatory gene expression of TNF-α (d), CD38 (e), and CD86 (f) were not induced in the IL-13 Mφs. Data were normalized to M0 Mφs and represent mean ± SEM. n = 12–14. Kruskal–Wallis test with Dunn’s correction. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.Additional file 3: Figure S2. IL-13 Mφs maintain their anti-inflammatory markers upon LPS stimulation. a–f Mφs were isolated from C57BL/6J mice. M0, M2, and IL-13 Mφs were left unstimulated (control) or were stimulated with LPS for 24 h. a–c qPCR showed that upon incubation with LPS, IL-13 significantly decreased the expression of the anti-inflammatory markers FIZZ1 (a) and CD206 (c) expression, whereas their expression of Ym1 (b) was maintained. Data were normalized to M0 Mφs and represent mean ± SEM. n = 8–9. d–f Pro-inflammatory gene expression of TNF-α (d), CD38 (e), and CD86 (f) were induced in the IL-13 Mφs upon LPS incubation as determined by qPCR. Data were normalized to M0 Mφs and represent mean ± SEM. n = 8–9. Kruskal–Wallis test with Dunn’s correction  or one-way ANOVA with a Bonferroni post hoc test . *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.Additional file 4: Figure S3. IL-13 Mφ transplantation does not affect astrogliosis or the Mφs/microglia presence at the lesion site. a–h Immediately following injury, C57BL/6J mice received vehicle, M2 Mφs or IL-13 Mφs. a, b Quantification of astrogliosis by GFAP intensity analysis (a) and Mφ/microglia presence by Iba-1 intensity analysis (b) showed no differences between treatment groups. Data are shown as mean ± SEM. n = 11–14 mice/group. c–h Representative images from the spinal cord sections are shown. All analyses were quantified within square areas of 100 μm × 100 μm perilesional placed as indicated in the figure, extending 600 μm rostral to 600 μm caudal from the lesion center (white line). Scale bar = 500 µm. Two-way ANOVA with Bonferroni post hoc test.Additional file 5: Figure S4. IL-13 Mφs are not present at the spinal cord 8 days after transplantation. a–d Immediately following injury, C57BL/6j mice received vehicle, GFP+M2 Mφs or GFP+IL-13 Mφs. a Quantification of the number of GFP+ cells at the injection, perilesional or lesion site after SCI. Data are shown as mean ± SEM. n = 3–6 mice/group. b–d Representative images of the lesion site from the spinal cord sections are shown. GFP+ cells are indicated by white arrows. Scale bar = 100 µm. Kruskal–Wallis test with Dunn’s correction.*P < 0.05, and **P < 0.01.Additional file 6: Figure S5. IL-13 Mφ transplantation does not affect the number of Iba-1+ cells at the lesion site. a Immediately following injury, C57BL/6j mice received vehicle, M2 Mφs or IL-13 Mφs. Quantification of the number of Iba-1+ cells at the lesion site showed no differences between vehicle- and IL-13 Mφ-treated mice. Data are shown as mean ± SEM. n = 8–9 mice/group. One-way ANOVA with Bonferroni post hoc test. *P < 0.05. n.s. not significant.Additional file 7: Figure S6. IL-13 Mφs secrete IL-13 and Arg1 Mφs express Arg1. a Mφs were isolated from IL-4R WT BALB/c mice. IL-13 secretion by the IL-13 Mφs was confirmed using ELISA. Data represent mean ± SEM. n = 2 independent in vivo experiments. b, c Mφs were isolated from C57BL/6J mice. Overexpression of Arg1 by the Arg1 Mφs compared to M0 Mφs was confirmed on gene  and protein  level. Data were normalized to M0 Mφs and represent mean ± SEM. Kruskal–Wallis test with Dunn’s correction. *P < 0.05, **P < 0.01, and ***P < 0.001.Additional file 8: Figure S7. IL-13 treatment does not influence cell survival of a murine or human neuroblastoma cell line. a, b The murine  and human  neuroblastoma cell lines express the alpha-1 subunit of the IL-13R and the alpha unit of the IL-4R as determined via immunocytochemistry. Scale bar = 50 µm. c, d Neuro2A (c) or SH-SY5Y (d) cells were treated with different concentrations of rIL-13  for 24, 48 or 72 h. Overall, IL-13 did not affect cell viability of both cell lines determined by an MTT assay. Data were normalized to untreated control and are shown as mean ± SEM. n = 4–5. e–h Neuro2A  or SH-SY5Y  cells were treated for 48 h with different concentrations of SNAP  to induce cell death. In addition, cells were co-treated  or pre-treated  for 24 h with different concentrations of rIL-13 . Using an MTT assay, neither co-treatment nor pre-treatment with IL-13 protected cells against cell death. Data were normalized to untreated control (= dotted black line) and are shown as mean ± SEM. n = 4–5. Kruskal–Wallis test with Dunn’s correction. *P < 0.05, **P < 0.01, and ***P < 0.001.Additional file 9: Table S2. IL-4 secretion. *BDL, below detection limit of ELISA kit (4 pg/ml).


In the crystal of the title hydrazide, the molecules are linked into (001) sheets by N—H⋯N and N—H⋯O hydrogen bonds. 10H12N2O2, are approximately coplanar with the exception of those at the ends: the terminal allyl carbon atom and terminal hydrazide nitrogen atom are displaced from the mean plane by 0.67 (2) and 0.20 (2) Å, respectively. In the crystal, the molecules are linked by N—H⋯O and N—H⋯N hydrogen bonds, which give rise a two-dimensional network propagating in the (001) plane.The non-H atoms of the title compound, C R—C(=O)—NH—NH2 functional group may act as a pharmacophore and present biological activity  Å, see Fig. 2The X-ray diffraction analysis revealed that the non-hydrogen atoms are approximately coplanar with the exception of the terminal atoms, which deviate by 0.67 (2) Å for C10 and 0.20 (2) Å for N2. In the crystal, the molecules are connected by N—H⋯O and N—H⋯N hydrogen bonds involving the carbohydrazide moieties of symmetry-related molecules Fig. 2 that forin vacuo. The resulting colourless oily mass was treated with hydrazine hydrate  and refluxed for 10 h in ethanol (40 ml). The reaction mixture was left overnight and colourless crystals suitable for X-ray characterization were obtained, filtered off and washed with ethanol. Yield: 7.0 g, (73%), melting point: 355–356 K.A mixture of ethyl-4-hydroxybenzoate  and allyl bromide  in acetone (100 ml) was refluxed for 20 h over anhydrous potassium carbonate . The filtrate was collected and the solvent removed −1): 1650 ν (C=Oester), 1621, 1575 ν (C=C), 3328, 3280, 3183 ν (NH—NH2).FT–IR (KBr), , δ: 7.72 , 6.94 , 7.65 , 4.13 , 5.42 , 5.32 , 6.05 , 4.58 .Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2414314622011956/hb4420sup1.cifCrystal structure: contains datablock(s) I, General. DOI: 10.1107/S2414314622011956/hb4420Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2414314622011956/hb4420Isup3.cmlSupporting information file. DOI: 2210836CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The additional Cu(ii) ions are coordinated to the nitrogen atoms of the triazole units. Nonzero out-of-phase signals are observed for the Tb derivatives (2a and 2b) and they exhibit frequency-dependent out-of-phase signals indicating a single-chain magnet behavior.A new nitronyl nitroxide, namely NIT-Ph- via single-crystal X-ray structural analysis.A new nitronyl nitroxide and two types of one-chain Cu–Tb complexes have been successfully synthesized, and the molecular structures have been elucidated  A number of metal complexes based on nitronyl nitroxides, including 2p–3d, 2p–4f heterobispin and 2p–3d–4f heterotrispin complexes, have been synthesized in recent years.6–8 Moreover, metal-radical complexes are good derivatives for studying magneto-structural correlation, which not only is important to understand the magnetic exchange interactions between the metal and the radical, but also for the design of novel nitronyl nitroxide radical ligands for the development of new molecular magnetic materials. To design more complexes with topological structures, special attention has been paid to functionalized radicals. Nitronyl nitroxide radicals that contain a triazole ring are good derivatives because these free radicals can bond to the metal ions via the triazole nitrogen atoms and the radical oxygen atoms, allowing for the formation of polynuclear clusters.9,10 Herein, a functional NITR containing triazole ring named NIT-Ph-p-OCH2trz methoxy)phenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, 5NIT-Ph-p-OCH2trz·0.5C6H14]n and [LnCu(hfac)5NIT-Ph-p-OCH2trz]n, were obtained. Complex 2a exhibits a ladder-type chain structure, while complex 2b features a 1D Ln chain bridged by the NO moieties of the radical ligands. Both 2a and 2b show frequency-dependent out-of-phase signals in a zero field with an oscillation of 3 Oe, indicating single-chain magnet behaviors.Paramagnetic nitronyl nitroxide radicals (NITRs) are promising building blocks for the synthesis of novel heterospin molecular magnetic materials.All reagents were purchased from commercial sources and used without further purification. Elemental analysis for C, H, and N was performed using a PerkinElmer elemental analyzer model 240. Powder X-ray diffraction (PXRD) data for all seven complexes were collected at room temperature on a Rigaku Ultima IV diffractometer using graphite monochromated Cu-Kα radiation. Magnetic measurements were measured on a SQUID VSM and PPMS-9 magnetometer. The magnetic susceptibilities were corrected for the diamagnetic contribution of the constituent atoms using Pascal's constants.p-OCH2trz was synthesized according to literature methods.11 Yield: 0.61 g (52%). Anal. calcd for C16H20N5O3(%): C, 58.17; H, 6.10; N, 21.20. Found: C, 58.24; H, 6.12; N, 21.26.The radical ligand of NIT-Ph-3·2H2O  was dissolved in 20 mL of dry boiling hexane and the solution was heated to reflux for 4 hours. Then, a solution of NIT-Ph-p-OCH2trz  in 3 mL of dry CHCl3 was added. The solution was refluxed for 30 minutes, and then solid Cu(hfac)2  was added. The resulting solution was stirred for 20 minutes and then cooled to room temperature and filtered. The filtrate was kept at room temperature; after two days, blue needle-like crystals  and green block crystals , suitable for X-ray diffraction were obtained using the evaporation method. Anal. calcd for C44H32CuF30N5O13Tb (2a) (%): C, 32.40; H, 1.98; N, 4.29. Found: C, 32.36; H, 1.95; N, 4.26. IR(KBr): 1651(s), 1501(m), 1474(m), 1256(s), 1226(s), 1142(s), 801(m), 662(m), 587(m) cm−1. Anal. calcd for C41H25CuF30N5O13Tb (2b) (%): C, 31.01; H, 1.59; N, 4.41. Found: C, 31.05; H, 1.61; N, 4.39. IR(KBr): 3247(s), 1706(s), 1610(s), 1482(m), 1261(s), 1222(s), 1163(s), 1007(s), 767(s), 727(s) cm−1.Tb(hfac)F2 with SHELXL-2014.12 All the non-hydrogen atoms were refined anisotropically and the hydrogen atoms were added geometrically and refined as riding atoms with a uniform value of Uiso. CCDC: 1973721 for 1, 1973720 for 2a and 1973722 for 2b.All the crystal structures were determined using a Rigaku Saturn CCD diffractometer employing graphite-monochromated Mo-Kα radiation at 113 K. The structures of the ligands and Tb complexes were solved by direct methods using the SHELXS-2014 program and refined by full-matrix least-squares on Pbca. The molecular structure is shown in b-axis displays a wavelike form.Compound 1 crystallized in the orthorhombic space group P1̄ group space. As shown in iii) ion is coordinated to one N atom from the triazole ring of the ligand, six O atoms from the three hfac ligands and one O atom from the NO group of the nitronyl nitroxide radical, resulting in a distorted square-antiprismatic geometry  and Tb–N distances are 2.346(5) and 2.578(6) Å, respectively, while the Tb–O(hfac) distances range from 2.327(4) to 2.363(3) Å. The Cu(ii) ion is in a {NO5} coordinate environment in which the axial positions are occupied by one O atom (O1) from the NO group and one N atom from the triazole ring of another molecule. The Cu–O and Cu–N bond distances are 2.408(5) and 2.467(6) Å in the axis positions, while the Cu–O bond lengths range from 1.941(6) to 1.953(5) Å. The paramagnetic organic ligand is linked to two Tb(iii) and two Cu(ii) ions via its two NO groups and two N atoms of the triazole ring in a tetradentate μ4-η1:η1:η1:η1 mode, resulting in a ladder-like arrangement in which the anisole moieties form the rungs of the ladder. The Tb⋯Cu separations through the NIT motif and triazole ring are 8.50 and 6.93 Å, respectively. The Tb⋯Tb and Cu⋯Cu distances across the rungs are 8.67 and 11.09 Å, respectively 5(NIT-Ph-p-OCH2trz)]2 and a [TbCu(hfac)5 (NIT-Ph-p-OCH2trz)] unit, which results in two crystallographically independent 1D chains in the crystal lattice. Compared to complex 2a, the linear chain in 2b is constructed from the radical ligand and Tb(iii) spin carriers, and the Cu(ii) ions are located on the side-chain. In complex 2b, each radical behaves as a tridentate ligand and binds two ions and one Cu(ii) ion in the μ3-η1:η1:η1 mode. The NO groups of the NIT-Ph-p-OCH2trz radical bridges two Tb(hfac)3 units to construct a 1D chain. Each Tb(iii) ion is coordinated to two O atoms from two radical ligands and six O atoms from three hfac ligands (iii) ions are located in distorted square-antiprismatic geometries  ions in 2b are all five-coordinated  between the radical ligands. According to the structure, the magnetic behavior of compound 1 can be treated as alternating 1D chains with S = 1/2. The magnetic data was analyzed using a theoretical expression1 deduced from the spin Hamiltonian Ĥ = −[J(Ŝ1 + Ŝ2) + αJ(Ŝ2 + Ŝ3)]. The experimental data was a good fit with J = −2.71 cm−1, g = 1.99, and α = 0.36. The negative value of J confirms the antiferromagnetic activity of the ligands.χ = |J|/kT; 0 ≤ α ≤ 0.4; A = 0.25; B = −0.062935 + 0.11376α; C = 0.0047778 − 0.033268α + 0.12742α2 − 0.32918α3 + 0.25203α4; D = 0.053860 + 0.70960α; E = 0.00071302 − 0.10587α + 0.54883α2 − 0.20603α3; F = 0.047193 − 0.0083778α + 0.87256α2 − 2.7098α3 + 1.9798α4The magnetic susceptibilities of complexes 2 were measured on polycrystalline samples. The phase purity of the samples was verified by comparing experimental and simulated PXRD pattern XRD analyses Fig. S4. The varχmT values are 12.10 and 11.58 cm3 K mol−1 at room temperature, slightly lower than the expected value of 12.57 cm3 K mol−1 for one uncorrelated Tb(iii) ion  plus one isolated Cu(ii) ion  and one radical . For 2a, the χmT value is constant from 300 K to 25 K, then the value sharply increases to a maximum of 13.60 cm3 K mol−1 at 4 K. The value decreases to 13.52 cm3 K mol−1 at 2 K. The increase in the χmT value at low temperatures confirms the ferromagnetic interactions between the Tb(iii) ions and the coordinated NO groups of the organic radicals, while the decrease in the χmT value is attributed to the crystal field effects of the Tb(iii) ion. For 2b, the value of χmT decreases slowly to a value of 10.52 cm3 K mol−1 at 25 K, and then steeply decreases to 9.94 cm3 K mol−1 at 2 K. This can be ascribed to the stronger crystal field effect of the Tb ion, which overwhelms the ferromagnetic radical-Tb contributions on the χmT curve.14 Both complexes 2a and 2b show linear regions ranging from 9 K to 26 K and 4 K to 9 K, and the values of Δξ are 1.04 K and 0.22 K, confirming the 1D Ising-like character of these complexes  = ln(ωτ0) + Δeff/kBT.15 The best fit produced τ0 ≈ 1.90 × 10−5 s and Δeff/kB ≈ 5.93 K for 2a and τ0 ≈ 6.38 × 10−6 s and Δeff/kB ≈ 3.84 K for 2b  susceptibility feature strong frequency-dependent phenomena , and its Tb complexes (2) were successfully obtained. Complexes 2a and 2b displayed two different 1D chains. In particular, 2a showed a rare ladder-like arrangement of alternating 2p–3d–4f spins with exchange interactions propagating along the rails. Both 2a and 2b exhibit a slow magnetic relaxation behavior below 3.0 K. This study illustrates that functionalized nitronyl nitroxide radicals are promising building blocks for synthesizing 2p–3d–4f heterotrispin systems.A new functional nitronyl nitroxide, namely NIT-Ph-There are no conflicts to declare.RA-010-D0RA00018C-s001RA-010-D0RA00018C-s002


H,3H-dithienopyrazine with CuI lead to the formation of a three-dimensional coordination polymer.The reaction of ligand 5,7-dihydro-1 H,3H-dithienopyrazine (L) with CuI lead to the formation of a three-dimensional coordination polymer, incorporating the well known [CuxIx]n staircase motif (x = 4). These polymer [Cu4I4]n chains are linked via the N and S atoms of the ligand to form the three-dimensional coordination polymer poly[tetra-μ3-iodido-tetracopper], [Cu4I4(C8H8N2S2)]n (I). The asymmetric unit is composed of half a ligand molecule, with the pyrazine ring located about a center of symmetry, and two independent copper(I) atoms and two independent I− ions forming the staircase motif via centers of inversion symmetry. The framework is consolidated by C—H⋯I hydrogen bonds.The reaction of ligand 5,7-dihydro-1 H,3H-dithienopyrazine (L), with silver(I) nitrate leads to the formation of a two-dimensional coordination polymer, with the silver atom coordinating only to the S atoms of the ligand so forming chains. The nitrato anion bridges two equivalent silver atoms and so generates the network structure  Å from the mean plane of the four C atoms (C1–C4). This is considerably more than in the silver(I) nitrate two-dimensional coordination polymer or the ligand itself  atoms are fourfold coordinate; the Cu⋯Cu distances are not considered as bonds. Hence, atom Cu1 has a fourfold CuSI3 coordination geometry with the fourfold index parameter τ4 = 0.91  atoms in I, the three-dimensional structure is consolidated by C—H⋯I hydrogen bonds . The majority form two-dimensional coordination polymers with the pyrazine ligand bridging the [CuxIx]n chains. For example, catena-[bis(μ3-iodo)dicopper(I)] (μ3-bis(6-methylpyrazin-2-ylmethyl)thioether-N,N′,N′′,S)(μ2-iodo)tricopper(I)]  atoms. Two of the copper atoms have CuNI3 fourfold coordination geometries with τ4 index parameters of 0.92 and 0.89. The third copper atom has a CuNSI2 fourfold coordination geometry with a τ4 index parameter of 0.73. In comparison, the τ4 index parameters for the two copper atoms in I are 0.91 and 0.85.There are less than 15 polymeric structures in the Cambridge Structural Database  Å. In RABBUS the copper(I) atoms coordinate to the pyrazine N atoms and the S atom of the ligand. Here, the Cu—Npyrazine bond lengths are 2.053 (4), 2.070 (4) and 2.071 (4) Å and the Cu—S bond length is 2.3574 (15) Å. In I, the Cu2—Niv and Cu1—S1 bond lengths of 2.059 (4) and 2.3393 (16) Å, respectively, are very similar to those sited above. The Cu—I bond lengths involving the [CuxIx]n staircase motifs are very similar in all three compounds; they vary from 2.6131 (15) to 2.6485 (15) Å in AGIYEU01, from 2.5993 (7) to 2.8348 (7) Å in RABBUS, and from 2.6418 (8) to 2.7142 (9) Å in I. The Cu⋯Cu distances in I, Cu1—Cu1i = 2.7355 (16) Å and Cu1—Cu2ii = 2.9030 (14) Å, are similar to those in AGIYEU01 [2.7562 (19) Å] and RABUSS .In AGIYEU01, the Cu—NH,3H-dithienopyrazine (L), have been reported  in CH2Cl2 (10 ml) was introduced into a 16 mm diameter glass tube and layered with MeCN (2 ml) as a buffer zone. Then a solution of CuI  in MeCN (5 ml) was added very gently to avoid possible mixing. The glass tube was sealed under an atmosphere of nitrogen and left in the dark at room temperature for at least 2 weeks, whereupon deep-orange plate-like crystals of the title compound, (I), were isolated in the buffer zone. Analysis for C8H8N2S2Cu4I4 (Mr = 958.14 g mol−1): calculated (%): C 10.03, H 0.84, N 2.92, S 6.69; found (%): C 10.12, H 0.80, N 2.82, S 6.67. The IR spectrum is shown in Fig. S1 of the supporting information.diffrn_reflns_laue_measured_fraction_full value (0.889) below minimum (0.95). The effect here appears to be limited if one compares the bond lengths in the structure of the pure ligand . As stated by Spek  I, Global. DOI: 10.1107/S2414314620004010/lh4055Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2414314620004010/lh4055sup3.pdfCSD search. DOI: Click here for additional data file.10.1107/S2414314620004010/lh4055sup4.tifThe IR spectrum. DOI: 1991908CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


II cations in a bis-pentadentate manner and the sixfold coordination sphere of each nickel(II) atom is completed by a water molecule.In the binuclear nickel(II) title compound, the ligand 2,2′,2′′,2′′′-{tetrakis(sulfanediyl)}tetraacetic acid coordinates two Ni 4L1H), with NiCl2 leads to the formation of a binuclear complex, ]tetrakis(sulfanediyl)}tetraacetato-κ5O,S,N1,S′,O′:κ5O′′,S′′,N4,S′′′,O′′′)bis[aquanickel(II)] heptahydrate, {[Ni2(C16H16N2O8S4)(H2O)2]·7H2O} (I). It crystallizes with two half molecules in the asymmetric unit. The complete molecules are generated by inversion symmetry, with the center of the pyrazine rings being located at crystallographic centres of inversion. The ligand coordinates two NiII ions in a bis-pentadentate manner and the sixfold coordination sphere of each nickel(II) atom (NiS2O3N) is completed by a water molecule. The complex crystallized as a hepta-hydrate. The binuclear complexes are linked by Owater—H⋯Ocarbonyl hydrogen bonds, forming layers parallel to the (101) plane. This layered structure is additionally stabilized by weak C—H⋯O hydrogen bonds. Further O—H⋯O hydrogen bonds involving binuclear complexes and solvent water molecules, together with weak C—H⋯S hydrogen bonds, link the layers to form a supramolecular framework.Reaction of the ligand 2,2′,2′′,2′′′-{tetrakis(sulfanediyl)}tetraacetic acid ( The complex crystallized as a hepta-hydrate. Selected bond lengths involving the nickel atoms of the two molecules are given in Table 1The ligand coordinates two NiI, binuclear nickel(II) complexes are linked by Owater—H⋯Ocarbonyl hydrogen bonds, forming layers parallel to the (101) plane  have been reported tetrakis(sulfanediyl)}tetraacetic acid  was dissolved in 50 ml of THF, then NaOH , dissolved in a minimum amount of water (a few ml) was added. The volume was increased to 100 ml adding THF and then the reaction was left to stir under reflux for 1 h. TBr  dissolved in 50 ml of THF, was then added dropwise using an addition funnel. The mixture was stirred under reflux for 6 h. After evaporation of the solvent, the mixture was dissolved in 50 ml of deionized water, and HCl (puriss.) was added dropwise until a clearly acidic pH was obtained. The mixture was then stirred at room temperature for at least 1–2 h. The yellow precipitate that slowly formed was filtered off and washed with a minimum amount of water and then with CHCl3. The solid obtained (4L1H) was dried in vacuo and was then recrystallized from methanol.4L1:Spectroscopic data for H1H-NMR: 4.13 ; 3.37 . 13C-NMR: 172.82 ; 150.01 ; 34.31 ; 33.26 .16H20N2O8S4, MW = 496.60 g/mol: Calculated (%) C 38.70, H 4.06, N5.64, Found (%) C 37.35, H 3.99, N 5.4.Analysis for CM + K]+; 519.00[M+Na]+; 497.02[M + H]+; 422.86, 407.04, 247.88.ESI-MS: 534.97·7H2O (I):Synthesis of complex [ and 4L1  were mixed together in 20 ml of degassed water. The mixture was left at 353 K under stirring and nitrogen conditions for 2.5 h. The mixture was then filtered and left to evaporate in air for two weeks, yielding purple needle-like crystals of complex I (m.p. 553 K decomposition).16H20N2Ni2O10S4)·7 (H2O), Mw = 772.10 g mol−1. Calculated (%) C 24.89, H 4.44, N 3.63. Found (%) C 28.17, H 3.90, N 4.18. Deviation due to the probable loss of water molecules of crystallization, for example, loss of five water molecules gives calculated (%) C 28.18, H 3.55, N 4.11.Analysis for  ν: 3364(s), 2921(m), 1713(m), 1575(s), 1404(s), 1237(m), 1208(m), 1155(m), 1137(m), 928(m), 704(m).IR  I, Global. DOI: 10.1107/S2414314621012955/im4014Isup2.hklStructure factors: contains datablock(s) I. DOI: 2126552CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


In the title molecular complex, the molecules form stacks consisting of aggregates with disordered 1-aminopyrene molecule surrounded by two 4,6-diacetylresorcinol molecules. Neighbouring stacks are linked by hydrogen bonds between the amine H atoms of the 1-aminopyrene molecule with the adjacent carbonyl oxygen atom of the 4,6-diacetylresorcinol molecule. 16H11N·2C10H10O4, at 150 K has been determined. The molecules form stacks consisting of aggregates with disordered 1-aminopyrene molecule surrounded by two 4,6-diacetylresorcinol molecules. Neighbouring stacks are linked by hydrogen bonds between the amine H atoms of the 1-aminopyrene molecule with the adjacent carbonyl oxygen atom of the 4,6-diacetylresorcinol molecule.The structure of the title molecular complex, C They are used in a variety of fields, including paper, textiles and the chemical, photographic, and electronic industries  and 0.072 (2) for N1 and N1ly Fig. 1.S(6) rings  Å is observed in this aggregate, with Cg1 being the centroid of C3–C8 ring of diacetylresorcinol and Cg2 the centroid of one of the aminopyrene rings, C11–C18. These aggregates are packed into stacks by π–π stacking interactions between 4,6-diacetylresorcinol molecules. Neighbouring stacks are linked by hydrogen bonds between the amino H atom of the 1-aminopyrene molecule with the adjacent carbonyl oxygen atom of the 4,6-diacetylresorcinol molecule, thus forming a three-dimensional network . The filtrate was left overnight upon which dark-brown rectangular co-crystals were formed, weight = 0.04 g, yield = 19.5%, m.p. = 418 K, m/z = 605.62 (C16H11N·2C10H10O4). 1H NMR  δ 12.75 , 8.406 , 8.251 , 7.992 , 7.992 , 7.958 , 87.915 , 7.880 , 7.367 , 6.392 , 6.314 , 2.661 .A solution of 1-aminopyrene  dissolved in 10 ml of ethanol was added dropwise to 4,6-diacetylresorcinol (DAR)  dissolved in 10 ml of ethanol, 3 drops of acetic acid were added, and the mixture was stirred for 15 min at room temperature and then for about 3 h under reflux. Yellow fiber-like crystals of the Schiff base ligand were separated. They were filtered off and washed with 4 ml of ethanol, weight = 0.021 g, yield = 7.12%, m.p. = 523 K, 6.B atom was constrained to have the same ADPs as the C atom to which it is bonded. N—H bond distances were restrained to a target value of 0.88 (2) Å, and the H—N—H and C—N—H bond angles were restrained to be similar to each other. Subject to these conditions the occupancy rates refined to 0.428 (2) and 0.072 (2). O-bound H atoms were refined with Uiso(H) = 1.5Ueq(O). C-bound H atoms were positioned geometrically (C—H = 0.9–0.98 Å) and refined as riding on their parent atoms with Uiso(H) = 1.2–1.5Ueq(C).Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989022005588/yk2169sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S2056989022005588/yk2169Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989022005588/yk2169Isup3.cmlSupporting information file. DOI: 2174187CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information: