T cell activation initiates protective adaptive immunity, but counterbalancing mechanisms are critical to prevent overshooting responses and to maintain immune homeostasis. The CARD11-BCL10-MALT1 (CBM) complex bridges T cell receptor engagement to NF-κB signaling and MALT1 protease activation. Here, we show that ABIN-1 is modulating the suppressive function of A20 in T cells. Using quantitative mass spectrometry, we identified ABIN-1 as an interactor of the CBM signalosome in activated T cells. A20 and ABIN-1 counteract inducible activation of human primary CD4 and Jurkat T cells. While A20 overexpression is able to silence CBM complex-triggered NF-κB and MALT1 protease activation independent of ABIN-1, the negative regulatory function of ABIN-1 depends on A20. The suppressive function of A20 in T cells relies on ubiquitin binding through the C-terminal zinc finger (ZnF)4/7 motifs, but does not involve the deubiquitinating activity of the OTU domain. Our mechanistic studies reveal that the A20/ABIN-1 module is recruited to the CBM complex via A20 ZnF4/7 and that proteasomal degradation of A20 and ABIN-1 releases the CBM complex from the negative impact of both regulators. Ubiquitin binding to A20 ZnF4/7 promotes destructive K48-polyubiquitination to itself and to ABIN-1. Further, after prolonged T cell stimulation, ABIN-1 antagonizes MALT1-catalyzed cleavage of re-synthesized A20 and thereby diminishes sustained CBM complex signaling. Taken together, interdependent post-translational mechanisms are tightly controlling expression and activity of the A20/ABIN-1 silencing module and the cooperative action of both negative regulators is critical to balance CBM complex signaling and T cell activation.The online version contains supplementary material available at 10.1007/s00018-022-04154-z. Antigen recognition by the TCR initiates an adaptive immune response. Assembly of the CARD11/CARMA1-BCL10-MALT1 (CBM) signalosome bridges TCR/CD28 co-stimulation to canonical NF-κB signaling pathways and activates the MALT1 protease to drive survival, expansion, differentiation and effector functions of activated T cells –6. In coA20 restricts autoimmunity and inflammation by acting as a pivotal negative regulator of many immune and inflammatory pathways . A20 defA20 restrains inflammation by counteracting TNFα and TLR-induced NF-κB signaling and cell death pathways –14. MechVarious co-factors are binding to the C-terminal ZnF region to modulate ubiquitin binding and E3 ligase activity of cellular A20 , 24. AmoUsing immunoprecipitation coupled with quantitative mass spectrometry (MS), we identified ABIN-1 as a CBM complex interaction partner in activated Jurkat T cells. We demonstrate that ABIN-1 and A20 cooperate by forming a negative regulatory module in T cells, which binds to the CBM signalosome to limit NF-κB and MALT1 protease activation following TCR/CD28 stimulation. We show that interdependent post-translational processes tightly co-balance the expression levels of A20 and ABIN-1, revealing that the combined action of both factors tunes the strength and the length of TCR signaling.Jurkat T cells were grown in RPMI-1640 medium supplemented with 10% fetal calf serum (FCS) and 100 units/ml penicillin plus 100 g/ml streptomycin (P/S). HEK293 and HEK293T cells were cultured in DMEM supplemented with 10% FCS and 100 U/ml P/S, and passaged when confluence exceeded 80%. Human CD4 T cells were grown in RPMI-1640 medium supplemented with 10% FCS (filtered), 100 U/ml P/S, 1% GlutaMax (100 ×), 1% Na-Pyruvate, 1% Non-essential amino acids, and 50 mM-Mercaptoethanol in the presence of IL-2. Cells were maintained at 37 °C in a water-saturated atmosphere supplemented with 5% CO2. Jurkat T cells were stimulated with 200 ng/ml Phorbol 12-myristate 13-acetate  and 300 ng/ml Ionomycin  (P/I) or anti-CD3 /anti-CD28  antibody (CD3/CD28) in the presence of anti-mouse IgG1  and IgG2a  . For stimulation of primary T cells, cells were transferred to anti-CD3 (0.5 µg/ml) pre-coated 96-well plates and stimulated in the presence of soluble anti-CD28 and anti-mouse IgG1/IgG2a as described above. Inhibitor treatment was done with 25 μM proteasome inhibitor MG132 , 1 μM MALT1 inhibitor MLT-985 [+ T cells were isolated from PBMC by positive selection with CD4-specific microbeads (Miltenyi Biotec) and were stimulated with plate-bound anti-CD3  and anti-CD28  for 3 days. Genes were depleted using Alt-R CRISPR-Cas9 system . In brief, crRNA and tracrRNA  were mixed with 1:1 ratio and incubated with Cas9 protein  for 20 min at RT to form RNP complex. 1 × 106 activated human CD4 T cells were electroporated and RNP complex were delivered into cells with Neon transfection system (Thermo Fisher Scientific) with the optimum program . Electroporated cells were then immediately incubated with RPMI-1640 medium supplemented with 10% FCS (filtered), 100 U/ml P/S, 1% GlutaMax (100 ×), 1% Na-Pyruvate, 1% Non-essential amino acids, and 50 mM-Mercaptoethanol in the presence of IL-2 (500 U/ml).Single-guide RNAs (sgRNAs) targeting ABIN-1 , A20 (5′-GAGGCAATTGCCGTCACCTG-3′), and HOIL-1 (combination of 5′-ATGGACGAGAAGACCAAGAA-3′ and 5′-TGTACCACGATCTGGCACTG-3′) were cloned into BbsI–linearized pX458 vector. Transfection of Jurkat T cells and generation of KO Jurkat clones by serial dilution has been described , 34–37. For stable expression of proteins in parental or KO Jurkat T cells, cells were transduced with lentiviruses expressing pHAGE-hΔCD2-T2A-A20-Flag-Strep-Strep or pHAGE-hΔCD2-T2A-ABIN-1-Flag-Strep-Strep WT or mutant (mt) constructs. Viral transduction has been performed as previously described . For traFor cytokine induction and analyses by flow cytometry and ELISA, primary human T cells were rested in T cell medium in absence of IL-2 for 24 h and stimulated with anti-CD3/CD28 as indicated above. For detection of intracellular cytokines by flow cytometry, stimulation was performed in presence of Brefeldin A  for 5 h. Cells were washed with FACS buffer (PBS + 3% FCS) and stained for 30 min at 4 °C with Fixable Viability Dye eFluor 780 . Cells were fixed in 2% PFA (15 min RT) and permeabilized in IC buffer (0.1% saponin in PBS) (15 min RT). Fc block  was performed for 7 min at RT. Cytokine staining was performed at 4 °C for 30 min in the dark using IL-2-APC (BD #554567) and TNFα-FITC (BD #554512) (both 1:100 in IC buffer). Cells were washed twice with IC buffer, resuspended in 100 μl FACS buffer and analyzed on an Attune Acoustic Focusing Cytometer. To determine secretion of IL-2, cells were stimulated for 24 h and analyzed using a human IL-2 ELISA kit according to the manufacturer’s instructions (Thermo Fisher Scientific #88-7025-88).7 Jurkat cells were lysed in 450 μl co-IP buffer ) supplemented with 1% SDS. Lysates were homogenized through a syringe (26G) and boiled for 10 min before being lysed for 20 min at 4 °C, followed by three times successive centrifugation and transfer of supernatant . Aliquots of supernatant were retained for Western blotting, the remaining supernatant was diluted tenfold with co-IP stock buffer without SDS or inhibitors, and subjected to immunoprecipitation. For detection of A20-triggered ABIN-1 ubiquitination or A20-triggered self-ubiquitination, HEK293 cells were transfected with Flag-A20 WT, OTU mt, ZnF4/7 mt, Flag-ABIN-1, HA-WT-Ub, HA-K63-Ub (K63-only) and HA-K48-Ub (K48-only) and lysed in 450 μl co-IP buffer containing 1% SDS supplemented with 10 mM N-ethylmaleimide  and 50 μM MG132. Cells have been lysed as described above. For co-IP, overnight immunoprecipitation at 4 °C was performed with 250 μl MALT1 antibody , 1 μg ABIN-1 antibody , 30 μl HA antibody  followed by 1–2 h incubation with 18 μl rec-protein G Sepharose 4B beads . After this incubation, samples were centrifuged and the pellet was washed three times with co-IP buffer without protease and phosphatase inhibitors. The supernatant was removed, 25 μl 2 × SDS-PAGE loading buffer was added, beads were boiled at 95 °C for 5 min and analyzed by Western blot.To determine ubiquitination of endogenous MALT1, 3–5 × 106 cells were harvested , washed with ice-cold PBS and resuspended in 100 µl high salt buffer , 1 mM MgCl2, 0.5 mM EDTA, 0.1 mM EGTA, 1% NP40, 1 mM DTT, 10 mM NaF, 8 mM β-Glycerophosphate, 300 µM NaVanadate, and protease inhibitor cocktail (Roche)). Cells were lysed on a shaker for 20–30 min at 4 °C, and centrifuged for 30 min at 21,000×g at 4 °C. 20 μl of supernatant was removed and stored at − 80 °C for EMSA analysis. Western blot and EMSAs were performed as previously described [1–3 × 10escribed . Western7 cells were lysed in 800 μl co-IP buffer rotating for 20 min at 4 °C and centrifuged at 21,000×g, 15 min, 4 °C. Aliquots of supernatant were retained for Western blotting. For co-IP, overnight immunoprecipitation at 4 °C was performed with 0.5 μg BCL10 antibody , 1 μg ABIN-1 antibody  or 30 μl HA antibody , followed by 1–2 h incubation with 18 μl rec-protein G Sepharose 4B beads . For Strep-PD (pull-down), cell lysates were incubated with 30 µl Strep-Tactin Sepharose  and rotated overnight at 4 °C. Beads were washed three times with ice-cold co-IP buffer without protease and phosphatase inhibitors. The supernatant was removed, 25 μl 2 × SDS-PAGE loading buffer was added, beads were boiled at 95 °C for 5 min and analyzed by Western blot.3–5 × 10To evaluate the hΔCD2 surface expression of infected cells, cells were stained with anti-CD2-APC antibody  at 4 °C in the dark for 20 min. To evaluate the NF-κB reporter gene activity of the EGFP reporter gene, cells were stimulated as described above, washed and resuspended in PBS, and analyzed on Attune Acoustic Focusing Cytometer. Data were analyzed using FlowJo software 10.7.1.Generation and application of biotin-labeled MALT1 activity-based probes (MALT1 ABPs) to precipitate and detect protease active MALT1 has been described previously . MALT1 s7 cells per sample were left untreated or stimulated for 20 min with P/I before lysis in 1% NP40 buffer , 10 mM Na-Pyrophosphate, 10 mM Na-Glycerophosphate, 1% NP40, 20 mM NaF, 1 mM EGTA, 1 mM EDTA, 1 mM DTT, 10% Glycerol and protease inhibitors). BCL10 IP  was performed overnight and binding to Protein G Sepharose 4B beads (Life Technologies) was performed for 2 h at 4 °C. After IP, beads were washed 2 × in 1% NP40 buffer and 2 × in 50 mM Tris–HCl (pH 7.5) buffer. On-bead digestion was performed overnight at 37 °C after re-suspending the beads in 2 M urea dissolved in 50 mM Tris–HCl (pH 7.5) buffer and adding trypsin. The generated peptides were cleaned using in-house prepared SDB-RPS (Empore) Stage Tips [For identification of BCL10 interaction partners, CK1α KO Jurkat T cells reconstituted with CK1α WT or D136N were used. 6 × 10age Tips  prior toTNIP1, TNFAIP3 and RPII genes are as follows : ABIN-1/TNIP1, 5′-GTTCAACCGACTGGCATCCAA-3′ and 5′-AGACGCACCCTCTTTGTTGC-3′; A20/TNFAIP3, 5′-CTGAAAACGAACGGTGACGG-3′ and 5′-CGTGTGTCTGTTTCCTTGAGCG-3′; RPII, 5′-GTTCGGAGTCCTGAGTCCGGATG-3′ and 5′-CCTGCCTCGGGTCCATCAGC-3′. For quantitative real-time PCR, the LightCycler 480 instrument (Roche) and Takyon qPCR Kit for SYBR Assays (Eurogentec) was used with a standard LightCycler protocol. RNA polymerase II (RPII) was used as an internal standard.Total RNA was isolated with RNeasy Kit (QIAGEN) and transcribed into cDNA using the Verso cDNA synthesis Kit (Thermo Fisher Scientific) according to the manufacturer’s guide. Primer sequences of the p values.Statistical analyses and data visualization were performed in GraphPad Prism version 8.0. Statistical details can be found in the figure legends, including the statistical tests used for calculating Casein kinase 1α (CK1α) activity is essential for CBM complex assembly in activated T cells . Thus, wWe focused on ABIN-1, because its binding to BCL10 was highly enriched upon stimulation and we could confirm inducible binding after anti-BCL10 IP and Western blotting following P/I and TCR/CD28 stimulation of Jurkat T cells RNAs targeting ABIN-1 or A20 by CRISPR/Cas9 technology. Transfection of ABIN-1 and A20 sgRNAs caused efficient ablation of ABIN-1 and A20 proteins in the pools of primary human CD4 T cells, respectively . On the protein level, expression of FS-tagged A20 or ABIN-1 was slightly above the endogenous levels, yielding a mild overexpression of the transduced constructs in all settings Fig. C, D. We Next, we assessed the effects of ABIN-1 and A20 reconstitution or overexpression on MALT1 protease activation. MALT1-ABP PD assays revealed that rescue of ABIN-1 or overexpression of A20 impaired CD3/CD28-induced MALT1 protease activity in ABIN-1 KO Jurkat T cells Fig. G. In conTo further prove that A20 binding is required for the function of ABIN-1, we compared the effects of ABIN-1 WT, A20-interaction mutant ΔAHD1 (Δaa 410–428) and UBAN-defective mutant D472N in counteracting NF-κB signaling Fig. A 26, 2727. DeletABIN-1 is unable to control CBM complex-dependent NF-κB signaling in the absence of A20. Thus, we wanted to investigate, if the N-terminal ubiquitin-hydrolyzing or the C-terminal ubiquitin binding functions of A20 are required for binding to ABIN-1 and silencing of T cell signaling. To this end, we expressed A20 mutant constructs with destructive mutations either in the OTU domain (C103A: OTU mt) or the C-terminal ZnF region (ZnF4/7 mt: C624/627/779/782A) . As part of a negative feedback loop, NF-κB-dependent transcriptional activation led to A20/TNFAIP3 mRNA induction in response to TCR/CD28 co-ligation, which was further augmented in ABIN-1 KO Jurkat T cells. Similar to IκBα, ABIN-1 degradation following 30 and 60 min of P/I stimulation in Jurkat T cells was impaired by MG132 treatment, demonstrating that both are prone to proteasomal degradation upon T cell stimulation Supplementary file2 (PDF 2270 KB)Below is the link to the electronic supplementary material.


Introduction Differentiation therapy is a promising strategy for cancer treatment. The translationally controlled tumor protein (TCTP) is an encouraging target in this context. By now, this field of research is still at its infancy, which motivated us to perform a large-scale screening for the identification of novel ligands of TCTP. We studied the binding mode and the effect of TCTP blockade on the cell cycle in different cancer cell lines. Methods Based on the ZINC-database, we performed virtual screening of 2,556,750 compounds to analyze the binding of small molecules to TCTP. The in silico results were confirmed by microscale thermophoresis. The effect of the new ligand molecules was investigated on cancer cell survival, flow cytometric cell cycle analysis and protein expression by Western blotting and co-immunoprecipitation in MOLT-4, MDA-MB-231, SK-OV-3 and MCF-7 cells. Results Large-scale virtual screening by PyRx combined with molecular docking by AutoDock4 revealed five candidate compounds. By microscale thermophoresis, ZINC10157406 (6-(4-fluorophenyl)-2-[(8-methoxy-4-methyl-2-quinazolinyl)amino]-4(3H)-pyrimidinone) was identified as TCTP ligand with a KD of 0.87 ± 0.38. ZINC10157406 revealed growth inhibitory effects and caused G0/G1 cell cycle arrest in MOLT-4, SK-OV-3 and MCF-7 cells. ZINC10157406 (2 × IC50) downregulated TCTP expression by 86.70 ± 0.44% and upregulated p53 expression by 177.60 ± 12.46%. We validated ZINC10157406 binding to the p53 interaction site of TCTP and replacing p53 by co-immunoprecipitation. Discussion ZINC10157406 was identified as potent ligand of TCTP by in silico and in vitro methods. The compound bound to TCTP with a considerably higher affinity compared to artesunate as known TCTP inhibitor. We were able to demonstrate the effect of TCTP blockade at the p53 binding site, i.e. expression of TCTP decreased, whereas p53 expression increased. This effect was accompanied by a dose-dependent decrease of CDK2, CDK4, CDK, cyclin D1 and cyclin D3 causing a G0/G1 cell cycle arrest in MOLT-4, SK-OV-3 and MCF-7 cells. Our findings are supposed to stimulate further research on TCTP-specific small molecules for differentiation therapy in oncology. ZIN10157406 (6-(4-fluorophenyl)-2-[(8-methoxy-4-methyl-2-quinazolinyl)amino]-4(3H)-pyrimidinone) revealed an LBE of -9.55 ± 0.00 kcal/mol and a pKi of 79.57 ± 0.02 nM. ZINC17879278 amino]-6-[(pyrimidin-2-ylsulfanyl)methyl]pyrimidin-4-ol) showed a lowest binding energy of -9.50 ± 0.01 kcal/mol and a Ki of 68.29 ± 0.01 nM. ZINC9419129 amino]-6-(3-methoxyphenyl)pyrimidin-4-ol) showed a lowest binding energy of -9.50 ± 0.01 kcal/mol and a Ki of 42.07 ± 0.00 nM. ZINC14096305 (Artesunate) was used as positive control [i of 53.67 ± 0.01 µM. Our in silico studies indicated that TCTP docked at the p53 binding site [The ol Table . ZINC180 control –26 and sE. coli K12 ER2566 harboring plasmid pTCTP01 was expressed and successfully purified according to a previously described protocol [For microscale thermophoresis, human seTCTP in protocol . The expin silico screening results. ZIN10157406 showed best in vitro binding with a binding affinity of 0.87 ± 0.38 µM.  . Artesunate decreased TCTP expression by 23.19 ± 8.47% (1 × IC50) and by 60.25 ± 2.08% (2 × IC50). At the same time, p53 expression increased to 167.69 ± 15.77% after application of (IC50) of artesunate and 191.87 ± 20.20% after application of 2 × IC50 artesunate.After 24 h incubation with ZINC10157406, the TCTP expression significantly decreased by 70.26 ± 6.11% at the IC50 of ZINC10157406 did not significantly change the H-score (152.25 ± 17.10). However, application of the IC50 significantly decreased the H-score to 80.10 ± 10.89 (p˂0.05) and application of 2 × IC50 decreased the H-score to 74.31 ± 8.85 (p˂0.05).After 24 h incubation with ZINC10157406, MOLT-4 cells were fixed onto a glass slide and incubated with an anti-TCTP antibody. Evaluation of the H-score to quantify the TCTP expression, was conducted by the Pannoramic Viewer software , and MDA-MB-231 cells, which have a low TCTP expression, as negative control. ZINC10157406 exerted growth inhibitory effects in all TCTP-positive cell lines after 72 h. For MOLT-4, MCF-7, and SK-OV-3, the IC50 values were about 20 times lower than of artesunate as known TCTP inhibitor. In MDA-MB-231 cells, no significant effect could be observed. ZINC10157406 decreased TCTP expression in MOLT-4 cells nearly three times more than artesunate. At the same time, p53 expression increased significantly (p˂0.05) if ZINC10157406 was applied. Artesunate as control decreased TCTP expression less strongly. Artesunate itself increased p53 expression, which explains this strong change [For our research, we conducted the experiments with MOLT-4-, MCF-7 and SK-OV-3 cells, which have a high TCTP expression according to the NCI-60 Human Tumor Cell Lines Screen  decreased the TCTP expression but p53 decreased even stronger confirming that the p53 binding site of TCTP was blocked by ZINC10157406. Although still statistically significant, the effects seen with artesunate as positive control were weaker than with ZINC10157406.For additional verification of binding of ZINC10157406 to the TCTP-p53 interaction site, we conducted a co-immunoprecipitation experiment. This is a powerful standard technique for the detection of highly sensitive protein-protein interactions , 47. TCTin silico and in vitro as novel ligand of TCTP. Our molecular docking results in silico were confirmed by flow cytometric studies, Western blotting, immunocytochemistry and co-immunoprecipitation. Hence, we were able to demonstrate that the interaction between TCTP and p53 can be disturbed by specific small molecule ligands. ZINC10157406 is a potent TCTP ligand and may act as valuable lead compound for further drug development with TCTP as treatment target in tumor reversion and cancer therapy.In conclusion, we were able to identify ZINC10157406


However, recommended low therapeutic activities (4–8 MBq) limit the clinical applicability of SPECT [225Ac imaging exist [Targeted alpha therapy (TAT) using  (mCRPC) . The 225ng exist , 4. Part225Ac radionuclide therapy using quantitative 225Ac SPECT. For a mCRPC patient (65 years), imaging of the abdomen was performed 24 h p. i. of 8.1 MBq 225Ac-PSMA-I&T on a Siemens Symbia Intevo T16 SPECT/CT , lower adjacent window , HEGP collimator, 16 projections/head, 128 × 128 pixel, 210 s/projection). Reconstruction was carried out via a MAP algorithm (30i1s) [225Ac image with the effective half-life information determined from a previous 177Lu-PSMA-I&T imaging sequence [RBE = 5/MBq for the left and right kidney, respectively, compared with 0.27 and 0.24 Gy/GBq for the preceding 177Lu cycle (6.2 GBq). A comparison with the pre-therapy 18F-PSMA-I&T PET/CT demonstrates that 225Ac SPECT imaging for this patient was able to locate a small lesion in the right hip. The 225Ac-absorbed dose was determined as 0.26 SvRBE = 5/MBq, compared with 0.35 Gy/GBq for 177Lu-PSMA-I&T.In this study, we would like to demonstrate the general feasibility of image-based dosimetry for  patient 5 years, 225Ac-PSMA-I&T, which provides further insights into theranostic approaches using TAT in mCRPC patients.Our analysis demonstrates the feasibility of dosimetry for


T) was isolated from a brine sample collected from one of the Anderton brine springs in the Cheshire salt district, located in Northern England. Phylogenetic analysis of the 16S rRNA gene sequence revealed a close proximity to Motilimonas eburnea (98.30 %), followed by Motilimonas pumila (96.62 %), the two currently described species within the genus Motilimonas. Strain MKS20T forms white-beige-pigmented colonies and grows optimally at 28–30 °C, in 1–3 % (w/v) NaCl and at pH 7–7.5. The strain was facultatively anaerobic and showed a broader range of carbohydrate use than other species in the genus Motilimonas. Q-8 was the sole respiratory quinone and the major fatty acids (>10 %) were summed feature 3 (C16 : 1 ω6c and/or C16 : 1 ω7c) and C16 : 0. The polar lipid profile included diphosphatidylglycerol, phosphatidylethanolamine, phosphatidyglycerol and several unidentified lipids. The G+C content of the genomic DNA was 44.2 mol%. Average nucleotide identity and DNA–DNA hybridization data were consistent with assignment to a separate species. Based on the phylogenetic and genomic-based analyses, as well as physiological and biochemical characteristics, we propose that strain MKS20T  represents a new species of the genus Motilimonas, with the name Motilimonas cestriensis sp. nov.A novel slightly halophilic Gram-stain-negative bacterial strain (MKS20 The Cheshire salt district (UK) has an industrial heritage of salt production dating back to the Romans (first–second centuries AD) who excavated the land for its abundant salt-rock . This inSaline biotopes such as these are inhabited by uniquely adapted halophilic and/or halotolerant micro-organisms, and thus have become sought-after for their taxonomic novelty and potential biotechnological applications . DespiteBacillus, Halomonas, Salinivibrio, Pseudoalteromonas, Marinobacter and Planococcus and many showed production of hydrolases and anti-microbial compounds. Noteworthy among these new isolates was a novel white-beige pigmented bacterial strain, designated MKS20T. The taxonomic position of strain MKS20T was investigated using 16S rRNA gene sequencing, with the results indicating that it was affiliated to Motilimonas, a genus incertae sedis within the order Alteromonadales (www.bacterio.net/-classifphyla.html) [Motilimonas eburnea, which was isolated from a marine sediment in China, and Motilimonas pumila, which was isolated from the gut of the sea cucumber Apostichopus japonicus [T could be distinguished from the other species within the genus Motilimonas. We therefore propose the name Motilimonas cestriensis sp. nov. for the species represented by strain MKS20T.During an astrobiological- and bioprospection-based study of the Anderton brine springs, several microbial strains were isolated . The majaponicus . Based oT was isolated from a brine sample collected from the Anderton brine springs, Cheshire, UK , which, at the time of sampling, had a salinity of 4.2 %, a pH of 7.4 and temperature of 14 °C. Aliquots of the sample were serially diluted and streaked on nutrient agar  supplemented with 5 % (w/v) NaCl (Melford) and incubated at 30 °C for 3 days. A white-beige circular colony was observed and purified via subculturing on the same medium before being preserved at −80 °C in nutrient broth  supplemented with 2 % (w/v) NaCl and 20 % (v/v) glycerol (Melford). After purification, standard cultivation was done on NA with salinity adjusted to 2 % (w/v) NaCl to reduce osmotic stress and increase growth rate. The type strains, M. eburnea YH6T (MCCC 1H00122T) and M. pumila PLHSC7-2T (MCCC 1K03522T), were obtained from the Marine Culture Collection of China (MCCC). For all experiments, the three strains were grown on NA supplemented with 2 % (w/v) NaCl at pH 7.5 and incubated at 30 °C, unless otherwise stated.Strain MKS20T was extracted using Griffith’s method [blast (www.ncbi.nlm.nih.gov) and EzTaxon-e (www.ezbiocloud.net) [T and M. eburnea was extracted and sequenced by MicrobesNG  using Illumina HiSeq 2500 apparatus with the 250 bp paired-end protocol and a minimum coverage of 30×. The Bioinformatics services of MicrobesNG trimmed the DNA reads using Trimmomatic 0.30 [de novo assembly using SPAdes version 3.8 [www.ezbiocloud.net/tools/contest16s) [T (accession number: MW130885) and M. eburnea (accession number: KR610526) obtained via Sanger sequenced were compared with the 16S rRNA gene from their respective genome sequence using NCBI blast to confirm genome authenticity before deposition via the services of GFBio [M. pumila PLHSC7-2T was obtained from NCBI (accession: QZCH00000000) [T was 4 796 511 bp, and the G+C content was 44.2 mol% (ENA accession: PRJEB39676). The genome was composed of 168 contigs with N50 and L50 values of 170 736 nt and 9, respectively. In comparison, the genome size of M. eburnea was 4 618 837 bp and the G+C content was 46.2 mol% (ENA accession: PRJEB39823). The genome was composed of 189 contigs with an N50 of 160 712 nt and an L50 of 10.Genomic DNA of strain MKS20s method . PCR amps method . PCR prooud.net) . The gentic 0.30 , performsion 3.8 , and annsion 3.8 . Genome test16s) , and therast Annotation Server (http://rast.theseed.org). Analysis of annotated subsystem features indicated the presence of features in strain MKS20T that were absent in M. eburnea and/or M. pumila. These included subsystems features of capsular and extracellular polysaccharides, osmotic regulation (ectoine biosynthesis), detoxification , metabolism of aromatic compounds (benzoate degradation) and carbohydrate metabolism, which corresponded with strain MKS20T’s wider metabolic range as observed experimentally , applying statistical ‘formula two’ to calculate high-41 scoring segment pairs and sequence identities [http://enve-omics.ce.gatech.edu/) [Digital DNA–DNA hybridization (dDDH) was performed using DSMZ’s Genome-to-Genome Distance Calculator  .T was extracted from the genomic DNA, the 16S rRNA gene length of MKS20T was 1585 bp (accession number: MT578061). This sequence, combined with those of M. pumila and M. eburnea, were used to recalculate the 16S rRNA gene sequence relatedness between the three strains of Motilimonas using NCBI blastn. arb-silva (www.arb-silva.de) [T (www.atgc-montpellier.fr/phyml/). The phylogenetic tree was then re-calculated using the best substitution model GTR+G+IT. The robustness of the phylogenetic tree was confirmed by comparing this tree with all trees calculated with arb using all available models.The full 16S rRNA gene sequence of MKS20a.de) [T . ModelTeT and M. pumila was 96.62 %, and the similarity between MKS20T and M. eburnea was 98.30 %, which is lower than the 98.7 % species threshold set by Stackebrandt and Eders [Motilimonas are indeed different species, according to the guidelines set by Meier-Kolthoff [T, M. eburnea and M. pumila was 44.2, 46.2, and 45.5 mol% respectively, the >1 mol% variation between strain MKS20T and the other two species is indicative of strain MKS20T belonging to a separate species [The 16S rRNA gene similarity between MKS20nd Eders . In addiKolthoff  (Table S species . This is species  (Table S3 (Sigma-Aldrich) for 14 days in an anaerobic jar containing a bioMérieux GenBag and anaerobic indicator strip.For phenotypic characterization, all three strains were grown on NA (or NB for growth experiments) supplemented with 2 % (w/v) NaCl, at pH 7.5, and incubated at 30 °C, unless otherwise stated. Cell morphology was examined after Gram-staining under a light microscope at ×1000 magnification succeeding calibration with a 100 µm microscope ruler. Cell motility was tested using the hanging drop method. Growth ranges and optima were measured via optical density in NB at a salinity of 0, 0.5, 1, 2, 3, 4, 5, 8, 10 and 12 % (w/v) NaCl; temperatures of 5, 10, 15, 20, 25, 28, 30, 33, 35, 37 and 40 °C; and at pH 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5 and 10.0. The media for pH experiments were buffered using 50 mM MES , HEPES , TAPS  or CAPSO  . The potCarbon source oxidation was determined using Biolog GEN III MicroPlates inoculated using Biolog Inoculation Fluid-A, supplemented with 2 % (w/v) NaCl, and incubated for 3 days following the manufacturer’s instructions. Carbon source acidification was determined using API 50CH test strips (bioMérieux), inoculated using 50 CHB/E medium (bioMérieux) supplemented with 2 % (w/v) NaCl and incubated for 3 days. Enzymatic and biochemical properties of the strains were analysed using API 20E and API ZYM test strips (bioMérieux), according to the manufacture’s protocols, with the modification of the inoculum being prepared with a 2 % (w/v) saline solution. Nitrate reduction was also tested as part of the API 20E test kit. The presence of catalase was tested by suspending a 48 h grown culture in a drop of hydrogen peroxide (Sigma-Aldrich), while the presence of oxidase was tested using a 24 h culture and one drop of oxidase reagent (bioMérieux). Amylase production was tested as described by Kumar . AntibioWhole-cell fatty acid profiles were determined at Fera Science Ltd Environmental Laboratories  according to the methods in Sasser , using 4T were motile, Gram-stain-negative, facultatively anaerobic, 0.5–1.0 µm wide and 1.3–3.0 µm long. Cells form rounded white-beige-pigmented colonies between 1.0–1.5 mm in diameter within 24 h of growth. Optimal growth occurred at 1–3 % (w/v) NaCl , 28–30 °C  and at pH 7.0–7.5 . Strain MKS20T was oxidase- and catalase-positive, but negative for nitrate reduction and amylase production. Strain MKS20T showed no anaerobic growth on NA with or without NaNO3 and also tested negative for nitrate reduction using the API 20E test. According to API 50CH test results, our strain produced acid from selected substrates, indicating fermentative capabilities (T were resistant to ampicillin (2 µg), penicillin (1.5 IU) and streptomycin (10 µg), but susceptible to chloramphenicol (10 µg), erythromycin (10 µg), cefoxitin (30 µg), sulphafurazole (100 µg), tetracycline (10 µg), apramycin (20 µg), carbenicillin (100 µg) and levofloxacin (5 µg). Additional biochemical and physiological characteristics of strain MKS20T and related Motilimonas species are summarized in Cells of strain MKS20bilities . Cells oT were summed feature 3 (C16 : 1 ω6c and/or C16 : 1 ω7c) and C16 : 0, fitting with the profiles observed for the other species within this genus. The minor fatty acids (<10 %) consisted of summed feature 2 (C14 : 0 3-OH and/or iso-C16 : 1), summed feature 8 (C18 : 1 ω6c and/or C18 : 1 ω7c), C14 : 0, C18 : 0 and C17 : 0, and showed higher variability across the genus. The fatty acid profile of strain MKS20T differs from other species of Motilimonas in their relative proportions of its components as well as the absence of minor fatty acids such as C12 : 0  of strain MKS20T represents the first described member of the genus Motilimonas isolated from a non-marine environment, thus extending its ecological reach.Strain MKS20T can be distinguished from other recognized species of the genus Motilimonas. We therefore propose that the strain represents a novel species, with the name Motilimonas cestriensis sp. nov.The results from the genotypic, physiological and chemotaxonomic comparison show that strain MKS20Motilimonas cestriensis .N-acetyl-β-glucosaminidase. Negative for the Voges–Proskauer test, nitrate reduction, indole production and production of H2S. Acid is produced from glycerol, d-mannitol, trehalose, d-glucose, d-fructose and d-mannose, but not from potassium 5-ketogluconate, cellobiose or lactose. Cells are able to oxidize dextrin, maltose, trehalose, gentiobiose, turanose cellobiose, d-fructose, N-acetyl-β-d-mannosamine, inosine, d-sorbitol, d-mannitol, glycerol, l-alanine, l-glutamic acid, l-serine, methyl pyruvate, l-malic acid, bromo-succinic acid, Tween 40, acetic acid and l-aspartic acid, but not propionic acid, l-histidine, glucuronamide, methyl β-d-glucoside, d-serine, or sucrose.Cells are facultatively anaerobic, Gram-stain-negative, motile rods (0.5–1.0×1.3–3.0 µm). Colonies are white-beige-pigmented, circular, rounded, moderately opaque and 1–1.5 mm in diameter when grown on NA at 30 °C for 24 h. Optimal growth at 28–30 °C , 1–3 % (w/v) NaCl  and at pH 7.0–7.5 . Cells are amylase-, lipase- (C14) and urease-negative, but positive for oxidase, catalase, esterase (C4), esterase lipase (C8), acid phosphatase, alkaline phosphatase, α-glucosidase and 16 : 1 ω6c and/or C16 : 1 ω7c) and C16 : 0. The polar lipid profile consists of phosphatidyglycerol, diphosphatidylglycerol, phosphatidylethanolamine and several unidentified components . Q-8 is the sole respiratory quinone. The genomic DNA G+C content of the type strain is 44.2 mol%.The major fatty acids (>10 %) are summed feature 3 (CT (=DSM 109936T=MCCC 1K04071T), was isolated from an inland brine spring in Anderton, within the Cheshire salt district (UK).The type strain, MKS20Click here for additional data file.


Though the combination of photodynamic therapy (PDT) and chemodynamic therapy (CDT) appears to be very attractive in cancer treatment, hypoxia and overproduced glutathione (GSH) in the tumor microenvironment (TME) limit their efficacy for further application.2O2) to form hydroxyl radical (·OH) for CDT. More importantly, the hyperthermia generated by APCZ upon 1064 nm laser excitation not only permitted NIR-II photothermal therapy (PTT) and promoted CDT, but also triggered the decomposition of AIPH to give toxic alkyl radical (·R) for oxygen-independent PDT. Besides, the PDA together with CuS greatly decreased the GSH level and resulted in significantly enhanced PDT/CDT in both normoxic and hypoxic conditions. The tumors could be completely eradicated after 14 days of treatment due to the prominent therapeutic effects of PTT/PDT/CDT. Additionally, the feasibility of APCZ as a photoacoustic (PA) imaging contrast agent was also demonstrated.In this work, a smart hypoxia-irrelevant free radical nanogenerator  was synthesized in situ via coating copper sulphide (CuS)-embedded zeolitic imidazolate framework-8 (ZIF-8) on the free radical initiator 2,2′-azobis[2-(2-imidazolin-2-yl)propane]-dihydrochloride (AIPH)-loaded polydopamine (PDA). APCZ showed promising GSH-depleting ability and near-infrared (NIR)-II photothermal performance for combined cancer therapy. Once internalized by 4T1 cells, the outer ZIF-8 was rapidly degraded to trigger the release of CuS nanoparticles (NPs), which could react with local GSH and sequentially hydrogen peroxide (HThe novel APCZ could realize the cooperative amplification effect of free radicals-based therapies by NIR-II light excitation and GSH consumption, and act as a contrast agent to improve PA imaging, holding tremendous potential for efficient diagnosis and treatment of deep-seated and hypoxic tumors.The online version contains supplementary material available at 10.1186/s12951-021-01003-2. Reactive oxygen species (ROS)-mediated photodynamic therapy (PDT) and chemodynamic therapy (CDT) have gained widespread attention in cancer theranostics –3. Howev2O2) with transition metal catalysts  dihydrochloride , 2,2′-Azinobis , glutathione , 5,5′-dithiobis (2‐nitrobenzoic acid) (DTNB) and methylene blue  were purchased from Aladdin Chemical Co. Ltd. Hydrogen peroxide , hydrochloric acid , nitric acid  and dimethylsulfoxide  were purchased from Sinopharm Chemical Reagent Co. Ltd., China. Copper chloride dihydrate , sodium sulfide nonahydrate , polyvinyl pyrrolidone , fetal bovine serum (FBS), Dulbecco’s Modified Eagle’s Medium (DMEM), Dulbecco’s phosphate-buffered saline (PBS), 2′,7′-Dichlorodihydrofluorescein diacetate , penicillin–streptomycin, trypsin, 3--2,5-diphenyltetrazolium bromide , 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI), calcein acetoxymethyl ester , propidium iodide (PI), hematoxylin and eosin (H&E) staining kit and one step terminal deoxynucleotidyl transferase uridine triphosphate (dUTP) nick end-labeling (TUNEL) apoptosis assay kit were purchased form Sigma-Aldrich. All the reagents were used without further purification and aqueous solutions were prepared with deionized (DI) water.Dopamine hydrochloride (98%), tris(hydroxymethyl)aminomethane , zinc nitrate hexahydrate (Zn(NO2O for further use.The AIPH-loaded PDA (AP) NPs were synthesized on the basis of a reported method with minor modifications . Briefly2O (10 mL) with sonication, followed by addition of PVP-CuS dispersion  and then aqueous Zn(NO3)2·6H2O solution  under vigorous stirring; aqueous 2-MIM solution  was then poured into the mixture and the reaction was kept for another 15 min. The resultant APCZ NPs were isolated by centrifugation  and washed with DI H2O for several times. The APZ (AIPH/PDA@ZIF-8) and PCZ (PDA@CuS/ZIF-8) were synthesized in analogous fashion for various comparison studies.The PVP-CuS NPs were obtained according to a reported method  but withMorphology was observed by transmission electron microscopy . Hydrodynamic diameters and zeta potentials were investigated by dynamic light scattering . Chemical composition was characterized by X-ray photoelectron spectroscopy (XPS). Crystal and chemical structures were examined by X-ray powder diffraction  and Fourier transform infrared  spectroscopies, respectively. Optical absorption spectra were obtained from an ultraviolet–visible–near infrared spectrophotometer . Element contents in sample dispersions were analyzed by inductively coupled plasma atomic emission spectroscopy .−1) of APCZ dispersions to a 1064 nm laser . To evaluate the effect of power intensity on temperature variations, 200 µg mL−1 of APCZ aqueous dispersion was separately irradiated by a 1064 nm laser at different power intensities  for 10 min. The changes in temperature as well as thermal images were recorded using an infrared thermal imager .The photothermal performance was investigated by exposing various concentrations (0–200 µg mL+˙) showed characteristic optical absorption ranging from 400 to 900 nm. Briefly, aqueous mixture (2 mL) containing AIPH (20 µg mL−1) and ABTS (20 µg mL−1) were heated at 37 or 44 °C for 2, 4, and 6 h, respectively, and the UV–vis absorption spectra were recorded for analysis. To evaluate the impact of GSH on the ·R formation, aqueous mixture (2 mL) containing AIPH (20 µg mL−1), ABTS (20 µg mL−1), and GSH (0.5 mM) were heated at 37 or 44 °C for 6 h, respectively, followed by spectroscopic investigations. The results were compared with those obtained by aqueous mixture (2 mL) of APCZ (400 µg mL−1), ABTS (20 µg mL−1) and GSH (0.5 mM) heated at 44 °C (pH 5.0) for different time periods. To monitor the NIR-II light-induced ·R production, 2 mL aqueous mixture of APCZ (1 mg mL−1), ABTS (50 µg mL−1), and GSH (0.5 mM) was first irradiated by 1064 nm laser (1 W cm−2) for 10 min, and then incubated at pH 5.0 for 1, 2, 3, 4, 5, 6 h. The supernatant was collected and the absorption spectra were obtained for investigation.ABTS was chosen to be the indicator for ·R generation as the ABTS free radical (ABTS−1) dispersions were separately added to aqueous GSH (50 µM) solutions and allowed to stand for 12 h. DTNB (25 µM) was then added and the pH values of the mixtures were adjusted to 7.4. After incubation for another 10 min, the absorbance was recorded by a UV–vis–NIR spectrophotometer. The consumption of GSH by CuS was measured with the use of aqueous CuCl2 solution. Briefly, aqueous CuCl2 (50 µM) and GSH (50 µM) solutions were first mixed for different time periods, then DTNB (25 µM) was added and the pH values of the mixtures were adjusted to 7.4. After incubation for another 10 min, the absorbance for the mixtures were recorded for interpretation.To determine the GSH depletion by AP and CuS, DTNB (25 µM) was first mixed with different concentrations of GSH (12.5–50 µM) in PBS buffer  for 10 min to establish a standard curve of GSH by collecting the absorbance at 412 nm. To monitor the consumption of GSH by AP, different concentrations of AP . After incubation for 1 h, the absorbance of the mixture was recorded by a UV–vis spectrophotometer. To further study the effect of reaction time and H2O2 or GSH concentration on the ·OH formation, the absorbances of the MB + H2O2 + Cu2+ + GSH + HCO3− group  at specific time points (10–60 min) were recorded. Subsequently, the degradation of MB was investigated after treatment with different concentrations of H2O2 (0–10 mM) or GSH (0.25–8 mM), where the other parameters were kept unchanged. Additionally, PVP-CuS was used instead of Cu2+ to induce MB degradation. The absorbances of MB + H2O2 + PVP-CuS + GSH + HCO3− group  at specific time points  were recorded. To evaluate the photothermal effects on the Fenton-like reaction, the MB + H2O2 + PVP-CuS + GSH + HCO3− group  was separately conducted at room temperature (25 °C), 37, 45 and 53 °C. After incubation for 5 min, the absorbances for the mixtures were recorded.MB was used as the indicator for the detection of ·OH generated from Cu−1) for cellular uptake experiment in accordance to our previous work  = 5 µg mLous work . After 12), 4T1 cells (1 × 105) were first seeded and cultured at 37 °C overnight, followed by incubating with (1) PBS, (2) blank, (3) AIPH, (4) APZ, (5) PCZ, (6) PCZ and (7) APCZ, respectively. After co-culturing for 4 h, group (2), (3), (6) and (7) were irradiated by 1064 nm laser . Subsequently, these seven groups were treated with DAPI (10 µg mL−1) and DCFH-DA (10 µg mL−1) for another 25 min, the cells were then washed and imaged by CLSM. Likewise, group (5), (6) and (7) were conducted once more in a hypoxic atmosphere (1% O2). Excitation wavelengths for DAPI and DCF were 405 and 488 nm, respectively. To compare the free radicals generated from each group, the [AIPH] or/and [CuS] were set to be 12.6 µg mL−1 and 11.4 µg mL−1 respectively.DCFH-DA was chosen to be the indicator for the detection of intracellular free radicals production as its deacetylated product (non-fluorescent DCFH) could react with free radicals to form fluorescent 2′,7′-dichlorofluorescein (DCF). Under normoxic condition (21% O6) were first seeded and cultured at 37 °C overnight, followed by incubating with (1) PBS, (2) blank, and different concentrations of (3) APZ, (4) PCZ, (5) PCZ and (6) APCZ, respectively. After co-culturing for 12 h, group (5) and (6) were irradiated by 1064 nm laser  and then underwent incubation for another 12 h. Cell viabilities were determined using a standard MTT assay . Four-week-old female Balb/C nude mice were injected with 4T1 cells (1 × 10−1) were first prepared and the PA signals were then determined under a 970 nm laser treatment . For in vivo PA imaging, APCZ dispersion (20 mg kg−1) was first intravenously injected into the 4T1 tumor-bearing mouse and the PA signals were recorded at specific time points .For in vitro PA imaging, APCZ dispersions with various concentrations  after injection of the sample dispersions (20 mg kg−1) for 10 h. In vivo photothermal performance of PCZ and APCZ were studied via recording the temperature changes of the tumor site and a PBS-treated group was set as control. Subsequently, the changes of tumor volume and body weight in each group were measured every 2 days for 14 days to assess the therapeutic efficacy. After the 2-week treatment period, mice were sacrificed and the tumor sections and major organs  were collected to estimate the tissue damage by H&E and TUNEL staining assay.To evaluate the in vivo anti-tumor effects, 4T1 tumor-bearing mice were randomized into six groups (n = 4 per group): (1) PBS; (2) 1064 nm Laser; (3) APZ; (4) APCZ; (5) PCZ + Laser; (6) PCZ + Laser. Photothermal-related experiments were conducted with 1064 nm laser . Statistical analysis was carried out by one-way ANOVA test with SPSS software . Statistical significance was set at *p < 0.05, **p < 0.01, ***p < 0.001.2+ ions and CuS; aqueous 2-MIM solution was then added to induce the formation of ZIF-8 layer with simultaneous embedment of CuS. Since TME has a lower pH than normal tissues, the pH-responsive ZIF-8 coating is able to prevent the premature leakage of both CuS and AIPH, thus ensuring its biosafety. Moreover, the CuS embedded in the ZIF-8 can endow remarkably enhanced NIR-II absorption for PTT as well as GSH-depleting capability for the APCZ to provide free radicals-mediated therapies.PDA, a nature-inspired biopolymer, is recognized as a new class of biomaterials for biomedical applications. In addition to being a popular photothermal agent for PTT, the abundant aromatic rings and functional groups make it possible to load chemical drugs and carry out further chemical modifications –37. On t2+ and Zn2+ . After loading with AIPH, the spectrum of AP presented the characteristic peaks of both AIPH and PDA. Besides, the peaks at 1640 and 620 cm−1 on the green curve were originated from the C=O in PVP and Cu–S in CuS respectively, indicating that the CuS was modified by PVP  = 25 µM. Figure S8. The degradation of AP in GSH, acid (pH 5.0) and acidic GSH (pH 5.0) for 12 h. [GSH] = 1 mM, [AP] = 100 µg mL−1. Figure S9. Detection of GSH (50 µM) depletion by aqueous CuCl2 (50 µM) solution for 10, 30, 60, 120, 180, 240 and 360 min, respectively. [DTNB] = 25 µM. Figure S10. UV–vis absorption spectra of PVP-CuS (25 µg mL−1) after incubation with various concentrations of GSH  for 6 h. Figure S11. Digital photos of PVP-CuS/GSH mixtures (separated by centrifugation and re-dispersed in 400 µL of DI H2O) after 6 h of reaction. [PVP-CuS] = 25 µg mL−1, [GSH] = (1) 0 mM, (2) 1 mM, (3) 2 mM, (4) 4 mM and (5) 10 mM. Figure S12. Relative Cu ions release from PVP-CuS/GSH mixtures after incubation for 6 h. [PVP-CuS] = 25 µg mL−1, [GSH] = 1, 2, 4 and 10 mM. Figure S13. TEM images of (a) PVP-CuS and (b-f) PVP-CuS/GSH mixtures after 24 h of reaction. [PVP-CuS] = 25 µg mL−1, [GSH] = 10 mM. Figure S14. Cumulative AIPH release profile of APCZ in PBS buffer of pH 7.4, pH 7.4 + GSH (10 mM), pH 5.0 and pH 5.0 + GSH (10 mM) for 24 h. Data shown as mean ± SD, n = 3 per treatment. Figure S15. Cumulative Cu ions release profile of APCZ in PBS buffer of pH 7.4, pH 7.4 + GSH (10 mM), pH 7.4 + GSH (10 mM) + Laser, pH 5.0, pH 5.0 + GSH (10 mM) and pH 5.0 + GSH (10 mM) + Laser. The orange arrows represented laser  treatment, each time point was radiated for 10 min. Data shown as mean ± SD, n = 3 per treatment. Figure S16. UV–vis−NIR absorption spectra of APCZ and APCZ/GSH mixture after incubation in aqueous ABTS solution (44 °C) for 6 h. [APCZ] = 400 µg mL−1, [GSH] = 0.5 mM, [ABTS] = 20 µg mL−1. Figure S17. IC50 of PCZ group in normoxic condition calculated from MTT results by GraphPad Prism 8 software. Figure S18. (a) The blood clearance kinetics of APCZ after intravenously administration. (b) Biodistribution analysis of APCZ in 4T1 tumor bearing mice after the tail vein injection for 24 and 48 h. Data shown as mean ± SD, n = 3 per treatment. Figure S19. Standard curves of (a) mouse TNF-α and (b) mouse IFN-γ. O.D. means optical density (absorbance at 450 nm). (c) TNF-α and (d) IFN-γ levels in sera isolated from different groups after 7-day treatments. Data shown as mean ± SD, n = 3 per treatment. Statistical significance was set at *p < 0.05, **p < 0.01, ***p < 0.001. Figure S20. H&E staining images of major organs after different treatments. Scale bars = 50 μm. Table S1. IC50 of different groups calculated from MTT results by GraphPad Prism 8 software.


The structures of the new compounds were elucidated by analysis of NMR spectroscopic spectra and HRESIMS data. The absolute configurations of 1 and 2 were established by spectroscopic data analysis and comparison of specific optical rotation, as well as the biosynthetic consideration. Additionally, compounds 1, 2, 4–6, and 8 showed significant nitric oxide (NO) production inhibition in Lipopolysaccharide (LPS)-induced BV-2 microglial cells with the IC50 values ranging from 9.06 to 14.81 μM.Two new sterpurane sesquiterpenoids named sterpurol D (The online version contains supplementary material available at 10.1007/s13659-021-00299-9. Endolichenic fungi parasitizing in the thalli of lichens bear resemblance to the endophytes residing in the tissues of higher plants –3. In thStereum purpureum in 1981 + . Its 1H and 13C NMR spectra . The location of the hydroxyl group at C-7 in 2 was determined by the observation of HMBC correlations from H-7 to C-6, C-8, C-9 and C-15. Detailed analysis of HSQC and HMBC spectra confirmed the structure of 2 as shown in Fig. 2 was determined by analysis of NOESY data. The obvious NOESY correlations  and H-17, indicated that they were all positioned on the same face of the tricyclic structure. In addition, the correlations of H-10 with Hb-9 (δH 1.71) indicated they were opposite orientation. The absolute configuration of 2 was determined using the modified Mosher's method + , three degrees of unsaturation. The 1H-NMR spectrum of 3 displayed resonances for one doublet and three singlet methyls at δH 0.96 , 1.16 , 1.22 , 1.69 , an olefnic proton at δH 5.30 , an oxymethine at δH 3.34  and other signals for aliphatic protons  located at C-9 of the bicarbocyclic ring moiety. The NOESY spectrum was measured in MicroCryoProbe . NOEs of 3-OH with Ha-1 and H-14, H-2 with Hb-1 and H-9, H-8 with H-10 assigned its relative configuration as shown , sterpurol B (5) D25 + 26.4 ; UV (MeOH) λmax (log ε) 230 (3.63); IR (KBr) νmax 3470, 2950, 2313, 1738, 1454, 1375, 1241, 1150, 1033, 647, 605 cm−1; 1H and 13C NMR data, see Table m/z 343.1876 [M + Na]+ .Sterpurol D : Colorle2): Colorless solid; [α]D25 + 758.8 ; UV (MeOH) λmax (log ε) 234 (3.34); IR (KBr) νmax 3388, 2933, 2871, 2316, 1726, 1451, 1373, 1246, 1121, 1026, 916, 792, 606 cm−1; 1H and 13C NMR data, see Table m/z 317.1704 [M + Na]+ .Sterpurol E (3): Colorless solid; [α]D25 – 47.8 ; 1H and 13C NMR data, see Table m/z 277.1775 [M + Na]+ .Cryptomaraone (5): Colorless solid; [α]D25 + 22.3 ; 1H and 13C NMR data, see Figs. S22 and S23; HR-ESI-MS m/z 301.1775 [M + Na]+ .Sterpurol B  and incubated for 24 h. Next, the cells were treated with the compounds (DMSO as solvent) at 20 μM for 24 h in DMEM with 1 μg/mL LPS. Cells treated with DMSO alone were used as the negative control. After adding 20 μL of 10 mg/mL MTT reagent to each well, the samples were shaken lightly and incubated at 37 °C for 4 h. The supernatant was removed, the blue-purple crystals were fully dissolved in DMSO (200 μL), and the absorbance of each well was read at 570 nm  [BV-2 murine microglial cells, acquired from Peking Union Medical College Cell Bank, were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% (v/v) heat-inactivated fetal bovine serum, penicillin (100 U/mL), and streptomycin (100 U/mL) in carbon dioxide cell incubator. When cell growth density outnumbered 90%, BV-2 cells were seeded in 96-well plates at a density of 2 × 10zerland) , 34. Per4 cells/100 μL of medium and incubated for 24 h. Then, cells were treated with 1 μg/mL of lipopolysaccharide (LPS) and various concentrations (0.1–20.0 μM) of test compounds (DMSO as solvent) for 24 h. An equal amount of DMSO and LPS were served as the controls; quercetin  was taken as the positive control). The NO concentration in the medium was measured by using a Nitric Oxide Assay Kit, according to the accumulated levels of nitrite in the supernatants by a standard Griess reaction [μL of the culture supernatant of BV-2 cells was reacted with 50 μL of Griess reagent I and Griess regent II successively in a 96-well plate. The absorbance at 570 nm of the mixture was measured using a microplate reader. IC50 values were calculated as the concentrations that reduced NO production by 50%. Quercetin was taken as the positive control.BV-2 cells were seeded into 96-well plates at 2 × 10reaction , 34. As α-Glucosidase Inhibitory assay was tested following the methods reported previously [μL) contained 572.4 μL of 0.05 M phosphate buffer (pH 6.8), 3.6 μL of enzyme solution (10 U/mL), and 36 μL of 0.4 mM inhibitors  were incubated at 37 °C for 10 min. Subsequently, 108 μL of 6 mM pNPG (4-nitrophenyl α-d-glucopyranoside) was added to the preincubated solutions, and the mixtures were incubated at 37 °C for 40 min. Then absorbance of the mixture at 405 nm was recorded. The negative control was prepared by adding PBS instead of α-glucosidase, the blank was prepared by adding solvent instead of tested compounds, and the inhibition rate was calculated as the following equation:eviously , 36 with1 − 8 were tested in vitro for antibacterial activity against nine bacteria . The tested bacteria were incubated in the beef extract-peptone medium (BPA) at 30 °C at 120 rpm for 12 h and the spore concentration was diluted to approximately 2 × 106 CFU/mL with BPA medium. 50 µL of suspension was added to 96-well microplates, then 50 µL of compounds (Ampicillin and streptomycin as positive control) dissolved in DMSO-BPA medium was added to give a final concentration of 100 µM. After incubation at 30 °C for 24 h, the absorbance of the mixture at 600 nm was recorded.Antibacterial activities were evaluated according to the previously published report  with sliSupplementary file 1 (DOCX 4069 KB)Below is the link to the electronic supplementary material.


Isolona dewevrei were investigated for the first time. Forty-seven oil samples were analyzed using a combination of CC, GC(RI), GC-MS and 13C-NMR, thus leading to the identification of 113 constituents (90.8–98.9%). As the main components varied drastically from sample to sample, the 47 oil compositions were submitted to hierarchical cluster and principal components analyses. Three distinct groups, each divided into two subgroups, were evidenced. Subgroup I−A was dominated by (Z)-β-ocimene, β-eudesmol, germacrene D and (E)-β-ocimene, while (10βH)-1β,8β-oxido-cadina-4-ene, santalenone, trans-α-bergamotene and trans-β-bergamotene were the main compounds of Subgroup I−B. The prevalent constituents of Subgroup II−A were germacrene B, (E)-β-caryophyllene, -6,12-oxido-elema-1,3,6,11(12)-tetraene and γ-elemene. Subgroup II−B displayed germacrene B, germacrene D and (Z)-β-ocimene as the majority compounds. Germacrene D was the most abundant constituent of Group III, followed in Subgroup III−A by (E)-β-caryophyllene, (10βH)-1β,8β-oxido-cadina-4-ene, germacrene D-8-one, and then in Subgroup III−B by (Z)-β-ocimene and (E)-β-ocimene. The observed qualitative and quantitative chemical variability was probably due to combined factors, mostly phenology and season, then harvest site to a lesser extent. The lipoxygenase inhibition by a leaf oil sample was also evaluated. The oil IC50 (0.020 ± 0.005 mg/mL) was slightly higher than the non-competitive lipoxygenase inhibitor NDGA IC50 (0.013 ± 0.003 mg/mL), suggesting a significant in vitro anti-inflammatory potential.The chemical variability and the in vitro anti-inflammatory activity of the leaf essential oil from Ivorian  Isolona Engl. is a genus belonging to the Annonaceae family and comprising about 20 species, widely distributed in tropical rain forests of West and Central Africa, and Madagascar. Five species of this genus grow wild in Côte d’Ivoire: I. cooperi, I. campanulata, I. soubreana, I. deightonii and I. dewevrei -5H-furan-2-one (1.2–7.8%) and massoia lactone (0.9–4.2%) -5H-furan-2-one and massoia lactone were not detected in I. dewevrei samples. The leaf oil from I. campanulata, on the other hand, was dominated by (E)-β-caryophyllene (8.2–30.5%), eudesm-5-en-11-ol (0.1–20.0%) and α-humulene (3.8–17.5%) [E)-β-caryophyllene (1.7–12.4%) was predominant in some I. dewevrei samples, its contents remained lower. In addition, eudesm-5-en-11-ol was completely absent from the leaf oil of I. dewevrei, while α-humulene was present in relatively small proportions (0.9–2.9%). Most of the other major constituents of I. dewevrei leaf oil were either absent or present at low contents in I. cooperi and I. campanulata leaf essential oils.The chemical composition of the leaf essential oil from nene 0.3–.7%, δ-3-8–17.5%) . AlthougI. dewevrei leaf essential oil (S44) was evaluated by determining its ability to inhibit lipoxygenases (LOX). Indeed, LOXs are nonheme iron-containing dioxygenases that convert linoleic, arachidonic and other polyunsaturated fatty acid into biologically active metabolites involved in the inflammatory and immune responses. Several inflammatory processes such as arthritis, bronchial asthma and cancer are associated with an important production of leukotrienes catalyzed by the LOX pathway from arachidonic acid [The in vitro anti-inflammatory potential of nic acid ,32,33,34I. dewevrei leaf essential oil to inhibit soybean lipoxygenase was determined as an indication of potential in vitro anti-inflammatory activity. I. dewevrei leaf essential oil exhibited an inhibition of LOX activity  were determined for the I. dewevrei leaf essential oil and for the non-competitive inhibitor of lipoxygenase, the nordihydroguaiaretic acid (NDGA)  is slightly higher than the IC50 value of NDGA (0.013 ± 0.003 mg/mL). The low ratio between the two values of IC50  makes it possible to consider this essential oil as a high inhibitor of the LOX activity, suggesting an in vitro anti-inflammatory potential [The ability of activity . The perd (NDGA) , usuallyotential .I. dewevrei shrubs at different phenological stages, at six sufficiently distant sites (Sites 1 to 6) of the Bossématié forest, Region of Abengourou, Eastern Côte d’Ivoire, and at two sites (Sites 7 and 8) of the Haut-Sassandra forest, Western Côte d’Ivoire. Geographical coordinates: Site 1 (6°31′34.7″ N and 3°28′15.0″ W), Site 2 (6°29′26.0″ N and 3°29′11.7″ W), Site 3 (6°27′44.9″ N and 3°32′25.5″ W), Site 4 (6°25′44.5″ N and 3°32′40.7″ W), Site 5 (6°26′07.2″ N and 3°28′27.2″ W), Site 6 (6°23′43.4″ N and 3°25′59.4″ W), Site 7 (6°53′40.2″ N and 6°55′36.3″ W) and Site 8 (6°54′52.7″ N and 6°57′21.1″ W). The harvest took place on one hand during the dry season  and during the rainy season on the other hand   . Plant mw/w). Plant material and essential oil extraction data are reported in n-pentane /diethyl ether  of increasing polarity . Seven fractions were eluted for each oil sample: F1 and F2 (eluted with n-pentane) contained hydrocarbons; F3–F6 (eluted with P/DE mixtures) contained medium polar compounds; F7 (eluted with diethyl ether) contained polar compounds. The respective weights of the fractions from the different samples are reported in the The essential oil samples were obtained by the hydrodistillation of fresh leaves for 3 h each, using a Clevenger-type apparatus. Yields were calculated from fresh material , equipped with a flame ionization detector (FID) and two fused-silica capillary columns , BP-1 (polydimethylsiloxane) and BP-20 (polyethylene glycol). The oven temperature was programmed from 60 °C to 220 °C at 2 °C/min and then held isothermal at 220 °C for 20 min; injector temperature: 250 °C; detector temperature: 250 °C; carrier gas: hydrogen (0.8 mL/min); split: 1/60; injected volume: 0.5 µL. Retention indices (RI) were determined relative to the retention times of a series of n-alkanes (C8–C29) with a linear interpolation (« Target Compounds » software from PerkinElmer). The relative response factor (RFF) of each compound was calculated according to the International Organization of the Flavor Industry (IOFI)-recommended practice for the use of predicted relative response factors for the rapid quantification of volatile flavoring compounds by GC(FID) . Methyl The essential oil samples and all fractions of chromatography were analyzed with a Clarus SQ8S PerkinElmer TurboMass detector (quadrupole), directly coupled with a Clarus 580 PerkinElmer Autosystem XL , equipped with a Rtx-1 (polydimethylsiloxane) fused-silica capillary column . The oven temperature was programmed from 60 to 230 °C at 2°/min and then held isothermal for 45 min; injector temperature, 250 °C; ion-source temperature, 250 °C; carrier gas, He (1 mL/min); split ratio, 1:80; injection volume, 0.2 µL; ionization energy, 70 eV. The electron ionization (EI) mass spectra were acquired over the mass range 35–350 Da.13C-NMR spectra were recorded on a Bruker AVANCE 400 Fourier transform spectrometer  operating at 100.623 MHz for 13C, equipped with a 5 mm probe, in CDCl3, with all shifts referred to via an internal TMS. The following parameters were used: pulse width = 4 µs (flip angle 45°); relaxation delay D1 = 0.1 s, acquisition time = 2.7 s for a 128 K data table with a spectral width of 25,000 Hz (250 ppm); CPD mode decoupling; digital resolution = 0.183 Hz/pt. The number of accumulated scans was 3000 for each sample or fraction .All 13C-NMR spectra of the samples and fractions with those of reference spectra compiled in the laboratory spectral library, with the help of a laboratory-made software [Identification of the individual components was carried out: (i) by a comparison of their GC retention indices on apolar and polar columns, with those of reference compounds ,37; (ii)software ,22. ThisI. dewevrei were submitted to a hierarchical cluster analysis (HCA) and principal component analysis (PCA) using XLSTAT software  [The chemical compositions of 47 leaf essential oil samples from  France) . Only coI. dewevrei leaf essential oil was evaluated by an in vitro lipoxygenase inhibition assay [The in vitro anti-inflammatory capacity of on assay ,42,43. Ton assay ,42,43.I. dewevrei leaf essential oil sample (S44) in dimethylsulfoxide (DMSO) were tested as the inhibitor solution for the LOX inhibition activity assay: 0.005, 0.010, 0.015 and 0.020 mg/mL.A stock solution of LOX was prepared by dissolving around 5.7 units/mL of LOX in PBS . Four concentrations of 0control is the activity of LOX in the absence of the inhibitor solution, and V0sample is the activity of LOX in the presence of the inhibitor solution [50 was calculated by the concentration of I. dewevrei leaf essential oil in DMSO inhibiting 50% of LOX activity.The LOX inhibition assays were performed by mixing 10 µL of LOX solution with 10 µL of inhibitor solution in 970 µL of boric acid buffer  and incubating them briefly at room temperature. The reaction started by the addition of 10 µL of substrate solution , and the velocity was recorded for 30 s at 234 nm. One assay was measured in the absence of the inhibitor solution, and one assay was measured with DMSO mixed with distilled water (99.85% of DMSO in distilled water) which made it possible to eliminate the inhibition effect of DMSO. No inhibitor effect of DMSO on the LOX activity was detected, and the LOX activity measured without inhibitor solution was considered as a control (100% enzymatic reaction). All assays were performed in triplicate. The percentage of lipoxygenase inhibition was calculated according to the equation:solution . The IC5I. dewevrei growing wild in Côte d’Ivoire was investigated through 47 oil samples. A combination of chromatographic ) and spectroscopic  techniques was used to determine the samples’ chemical compositions. One hundred and thirteen constituents accounting for 90.8–98.9% of the whole sample compositions were identified, and the main components varied drastically from sample to sample. Therefore, the 47 oil compositions were submitted to hierarchical cluster and principal components analyses, which evidenced three distinct chemical groups, each dividing into two subgroups. The Subgroup I−A was dominated by (Z)-β-ocimene, β-eudesmol, germacrene D and (E)-β-ocimene, while (10βH)-1β,8β-oxido-cadina-4-ene, santalenone, trans-α-bergamotene and trans-β-bergamotene were the main compounds of Subgroup I−B. The prevalent constituents of Subgroup II−A were germacrene B, (E)-β-caryophyllene, -6,12-oxido-elema-1,3,6,11(12)-tetraene and γ-elemene. The Subgroup II−B displayed germacrene B, germacrene D and (Z)-β-ocimene as the majority compounds. Germacrene D was the most abundant constituent of Group III, followed in Subgroup III−A by (E)-β-caryophyllene, (10βH)-1β,8β-oxido-cadina-4-ene, germacrene D-8-one, and then in Subgroup III−B by (Z)-β-ocimene and (E)-β-ocimene. Compounds bearing the eudesmane skeleton characterized Subgroup I−A and also Subgroup III−B to a lesser extent. Likewise, santalane, bergamotane and bisabolane skeletons were markers of Subgroup I−B, while the elemane skeleton was specific to Group II. Group III markers were oxygenated germacrane and cadinane compounds. Although genetic factors could not be completely excluded, the observed qualitative and quantitative chemical variability of the leaf essential oil from I. dewevrei could be related to mostly phenology and season, then harvest site to a lesser extent. A leaf oil sample (S44) was tested for its lipoxygenase inhibition ability. The oil IC50 value (0.020 ± 0.005 mg/mL) was slightly higher than the non-competitive lipoxygenase inhibitor NDGA IC50 value (0.013 ± 0.003 mg/mL). Therefore, this leaf essential oil exhibited significant in vitro anti-inflammatory potential.The chemical variability of the leaf essential oil from


PS/PSEN) genes encoding the catalytic components of γ-secretase accelerate amyloid-β (Aβ) and tau pathologies in familial Alzheimer’s disease (AD). Although the mechanisms by which these mutations affect Aβ are well defined, the precise role PS/γ-secretase on tau pathology in neurodegeneration independently of Aβ is largely unclear. Here we report that neuronal PS deficiency in conditional knockout (cKO) mice results in age-dependent brain atrophy, inflammatory responses and accumulation of pathological tau in neurons and glial cells. Interestingly, genetic inactivation of presenilin 1 (PS1) or both PS genes in mutant human Tau transgenic mice exacerbates memory deficits by accelerating phosphorylation and aggregation of tau in excitatory neurons of vulnerable AD brain regions . Remarkably, neurofilament (NF) light chain (NF-L) and phosphorylated NF are abnormally accumulated in the brain of Tau mice lacking PS. Synchrotron infrared microspectroscopy revealed aggregated and oligomeric β-sheet structures in amyloid plaque-free PS-deficient Tau mice. Hippocampal-dependent memory deficits are associated with synaptic tau accumulation and reduction of pre- and post-synaptic proteins in Tau mice. Thus, partial loss of PS/γ-secretase in neurons results in temporal- and spatial-dependent tau aggregation associated with memory deficits and neurodegeneration. Our findings show that tau phosphorylation and aggregation are key pathological processes that may underlie neurodegeneration caused by familial AD-linked PSEN mutations.Mutations in the presenilin (The online version contains supplementary material available at 10.1186/s40478-021-01259-7. Alzheimer’s disease (AD) is the most prevalent and disabling memory disorder in the elderly. AD is characterized by neurodegeneration, brain inflammation and accumulation of amyloid-β (Aβ) plaques and neurofibrillary tangles (NFTs) containing bundles of paired helical filaments (PHF) formed of aggregated hyperphosphorylated microtubule-associated protein tau (MAPT) in memory brain regions , 62. Phopresenilin (PSEN/PS) genes encoding presenilin 1 (PS1) and presenilin 2 (PS2) cause early-onset familial AD presumably by accelerating Aβ and/or tau pathologies [knockin mice [PS conditional knockout (cKO) mice [Autosomal dominant mutations in the hologies . PS are hologies , 63, 71.hologies , 50, 71.hologies , 16, 54,kin mice , 72. Conkin mice , a loss-kin mice . In agreKO) mice , 57, 70.Tau and lacking conditionally PS1  or both PS  genes. We found that partial PS inactivation in Tau transgenic mice results in accelerated phosphorylation and aggregation of tau and NF light chain (NF-L) in memory neural circuits coinciding with loss of synaptic proteins and exacerbated memory deficits.Here, we investigated the role of PS in tau pathology by examining in vivo the effects of partial or total loss of PS in postmitotic neurons of novel tauopathy mouse models expressing a FTD-linked human PS2 embryonically and PS1 specifically in forebrain glutamatergic neurons were previously described [PS1 f/f; PS2+/+ or PS1 f/f; PS2+/-; f: floxed), PS1 cKO  and PS cKO  mice were obtained by crossing floxed PS1/PS2-/-  or PS2±  males to heterozygous PS1 cKO; PS2± females . We generated novel non-transgenic control (PS1 f/f), tau , PS1 cKO;Tau  and PS cKO;Tau  mice by crossing PS1 cKO or PS cKO with Tau P301S transgenic mice . Tau P301S mice express the FTD-linked P301S Tau under the neuron-specific prion protein promoter [PS cKO mice (C57BL/6/129 background) lacking promoter . All expTail suspension test was performed by suspending the mouse from the tail for 5 min. Measures of movement of hind/forelimbs and clasping behavior are measured according to the severity of the hindlimb clasping . Hippocampus-dependent spatial memory was examined in the Morris water maze test as described . Mice weFor contextual fear conditioning, mice handled for three days (3 min/day) were placed in a conditioning chamber  for 3 min, foot-shocked (1 s/1 mA) and retained in the chamber for 2 min (immediate freezing) . Fear me3VO4, 1 mM EDTA, 1 mM ethylene glycol tetraacetic acid) containing protease and phosphatase inhibitors and boiled at 100 °C [3, 1 mM MgCl2, 0.5 mM CaCl2, 1 mM NaF and phosphatase and protease inhibitors). The homogenate was centrifuged  and the pellet (P1) was re-extracted with Buffer A and centrifuged . The supernatants were combined and centrifuged . The pellet was resuspended in ice-cold Buffer B  and loaded onto the top of a 0.85 M, 1 M and 1.2 M discontinuous sucrose gradient and centrifuged . The synaptosome fraction, collected from the 1 M–1.2 M sucrose interface, was diluted in equal volume of Buffer C . The suspension was spin at 32,800 × g for 1 h to obtain the presynaptic (supernatant) and the postsynaptic (pellet) fractions, the latter was resuspended in cold-ice Buffer D . Proteins were quantified using the protein assay kit (Invitrogen), resolved on SDS–polyacrylamide gel electrophoresis and detected by Western blotting using tau and NF antibodies  were deparaffinized in xylene, rehydrated and microwave heated in citrate buffer . Sections were incubated overnight at 4 °C with the indicated primary antibodies (Table For Congo Red staining we used the Amyloid Stain Congo Red (Sigma-Aldrich). Briefly, after deparaffinization and hydration, slides were stained in Mayer’s hematoxylin solution (Sigma-Aldrich) for 10 min and rinsed in tap water. Slides were placed in alkaline NaCl and stained with Alkaline Congo Red Solution (20 min). Samples were rinsed (× 3) in 100% ethanol, cleared in xylene, mounted in DPX Mountant (Sigma-Aldrich) and observed in Nikon Eclipse 90i microscope.2 optical windows . μFTIR study was carried out at the MIRAS beamline at ALBA Synchrotron Light Source  using a Hyperion 3000 microscope equipped with a 36 × magnification objective coupled to a Vertex 70 spectrometer (Bruker) and a mercury cadmium telluride (MCT) detector. Spectra acquisition and analysis were performed as reported [−1 spectral resolution, 10 μm × 10 μm aperture dimensions, and 128 scans. The measuring range was 4000–600 cm−1 and zero filling was performed with fast Fourier transform. Background spectra were collected from a clean area of each CaF2 window every 10 min. For each animal, a total of 100 spectra per cerebral region with a step size of 30 μm × 30 μm were acquired in the retrosplenial cortex and the corpus callosum. The spectra were obtained by means of Opus 7.5 software (Bruker).Mice were intracardially perfused for 10 min with 4% paraformaldehyde in 0.1 M Tris buffer (pH 7.4), brains were post-fixed in the same solution for 4 h at 4 °C, cryoprotected with 30% sucrose solution in 0.1 M Tris buffer for 48 h at 4 °C, and frozen in ice-cold 2-methylbutane (Sigma-Aldrich). Coronal Sections (8 μm) were cut on a cryostat (CM3050S Leica) and mounted onto polished CaFreported . Briefly−1 and 1656 cm−1, respectively. Intermolecular β-sheet structures corresponding to β-sheet aggregates absorb at 1625 cm−1. Antiparallel β-sheets corresponding to oligomeric β-aggregates were detected at 1695 cm−1. In addition, protein/lipid (Amide I/CH2) and lipid oxidation, determined by carbonyl content (C=O) absorbances at 1741 cm−1 and the unsaturated olefinic group (C=CH) at 3014 cm−1, were measured to provide biochemical composition of the brain tissue. To account for variations in the tissue thickness, absorbances values were normalized by the major lipid content (CH2) or the major protein content (Amide I), giving rise to the following ratios: β-sheet/α-helix (d2A1635/d2A1656), β-intermolecular/Amide I (d2A1625/d2A1635+1656), β-antiparallel/Amide I (d2A1695/d2A1635+1656), Amide I/CH2 (d2A1635+1656/d2A2921), C=O/CH2 (d2A1741/d2A2921) and C=CH/CH2 (d2A3014/d2A2921).Unscrambler X software (CAMO Software) was used for data processing. Second spectra derivative was calculated using a Savitsky-Golay algorithm with an eleven-point smoothing filter and a polynomial order of 3. Intramolecular β-sheet and α-helix protein secondary structures were detected by infrared absorptions at 1635 cmt-test when only two groups were compared. P values less than 0.05 were considered significant. The significance level is indicated using asterisks: * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001.Statistical analysis  was performed using one or two-way Analysis of Variance (ANOVA) followed by Sidak’s (age comparison) or Tukey’s post hoc test for multiple comparisons and −/−  mice, in which both PS are deleted in excitatory neurons of the postnatal forebrain [P < 0.0001). Interestingly, weight of the whole brain, cortex and hippocampus are significantly reduced in PS cKO mice at 6–12 months of age  and PS1 cKO;PS2orebrain . PS cKO  f/f and −/−, PS1 cKO, and PS cKO mice at 9 months of age. Biochemical analysis using anti-tau antibodies for phosphorylated Ser 202 (CP13) and Ser 202/Thr205 (AT8) present in early (pre-tangles) and advanced NFTs, and Ser 396/404 (PHF-1), a marker of late NFT stages in AD brain (Table P < 0.05), but not in the cortex, whereas post-hoc analysis revealed enhanced phosphorylated tau levels in PS cKO mice , PS1 cKO (1.44 ± 0.11/0.98 ± 0.21), and PS cKO (1.08 ± 0.14/1.15 ± 0.44) mice  and Iba1 (P < 0.01) levels indicating differences among groups. Post-hoc analysis revealed significant enhanced GFAP and Iba1 only in PS cKO mice (P < 0.05). These results demonstrate that phosphorylated tau is present in neurons and glial cells of PS cKO mice during inflammation and neurodegeneration.To study whether increased tau phosphorylation in glial cells was accompanied by inflammatory responses, we next examined astrocytic (GFAP) and microglial (Iba1) markers in the hippocampus of WT, PS2age Fig. b. Quanti and PS cKO;Tau  mice obtained by crossing PS1 cKO and PS cKO mice with mutant Tau transgenic (PS19) mice expressing the FTD-linked P301S mutation in excitatory neurons [;Tau mice compared to Tau mice   Fig. . In addiP < 0.05), restrosplenial cortex , entorhinal cortex  and basolateral amygdala  . Immunostaining with MC1 antibody showed neuronal somatodendritic aggregated tau staining in different brain regions, including the hippocampus, EC and basolateral amygdala in 6 month-old PS1 cKO;Tau and PS cKO;Tau mice , EC (P < 0.0001) and basolateral amygdala (P < 0.0001) , EC (P < 0.001) and amygdala (P < 0.01)  in AD brain  Fig. , indicat01) Fig. . These rPSEN1 mutations elevate levels of NF light chain (NF-L) in cerebral spinal fluid (CSF) of AD patients [P < 0.05; Fig. P < 0.0001; PS effect, P < 0.001; tau x PS interaction effect,P < 0.001;Fig. PS1 regulates the assembly of neurofilaments (NF) and patients , 71. To 2 and C = CH/CH2) and protein/lipid ratio (Amide I/CH2), but without significant changes among genotypes , intermolecular (d2A1625/d2A1635+1656) and antiparallel (d2A1695/d2A1635+1656) protein structures corresponding to non-aggregated, aggregated and oligomeric β-sheet structures, respectively [P < 0.05; area effect, P < 0.0001) and antiparallel  β-sheet structures were significantly higher in the CC of PS cKO;Tau mice , ruling out the possibility of motor and visual deficits. Although no differences were detected in the first trial, there were significant latency changes depending on genotype and trial . Tau, PS1 cKO;Tau and PS cKO;Tau mice exhibited significantly longer latencies that control mice . In the probe trial on day 6, PS cKO;Tau mice spent more time to find the target quadrant, and crossed less often and spent less time in the target quadrant than the rest of groups  and time (P < 0.001) . These results indicate PS dosage-dependent effects on hippocampal-dependent spatial and associative memory in Tau transgenic mice.To analyze the effects of tau pathology on hippocampal-dependent memory, we next tested 6 month-old littermate control, Tau, PS1 cKO;Tau and PS cKO;Tau mice in the Morris water maze (MWM) and contextual fear conditioning (CFC) tasks. Swimming speed during training in the visible platform test were similar among all groups (01) Fig. e. Post-ht-test: synaptosome, P < 0.05; presynaptic, P < 0.05; postsynaptic P < 0.05), and decreased PSD95 and CRTC1  and synaptophysin  in synaptic compartments of Tau transgenic mice  were decreased in the hippocampus of PS cKO;Tau mice coinciding with enhanced phosphorylated/total tau levels  is barely detected in neurons , microglia , oligodendrocytes  and astrocytes  in brain sections of control (WT) mice. Insets: magnified images of the indicated selected regions of CA3, CA1, CC and DG (upper images) are shown at the bottom. Abbreviations: CA1/CA3 hippocampus; DG: dentate gyrus; CC, corpus callosum.Additional file 2. NF staining and synchrotron-based µFTIR analysis of PS-deficient Tau mice. A, Immunohistological images of SMI312 (NF-H/M) in the basolateral amygdala of WT, PS1 cKO, PS cKO, Tau, PS1 cKO;Tau and PS cKO;Tau mice at 6 months of age. Insets: magnified images of the indicated left square regions showing prominent NF-H/M somatic staining in amygdalar neurons of PS cKO, PS1 cKO;Tau and PS cKO;Tau mice. Scale bar = 100 m. B, Representative average infrared spectra of WT and PS cKO;Tau mice in the retrosplenial cortex (RSC) and corpus callosum (CC) indicating the main absorptions peaks with their corresponding chemical functional groups. Abbreviation: a.u.: arbitrary units. C, Synchrotron-based µFTIR analysis of the RSC and CC of WT and PS cKO;Tau mice at 6 months of age showing lipid oxidation by C=CH/CH2 (d2A3014/d2A2921) and C=O/CH2 (d2A1741/d2A2921) ratios, and protein/lipid amount by Amide I/CH2 (d2A1635+1656/d2A2921) ratio. Values represent the minimum, the maximum and the median of the average of 100 spectra/mouse (n = 4 mice/group).Additional file 3. Phosphorylated tau is present in synaptosomes of Tau transgenic mice. A, Western blot images of phosphorylated tau and synaptic proteins in lysates and purified synaptosomes and presynaptic and postsynaptic fractions from hippocampus of 6-9 month-old WT and Tau mice. B-D, Quantitative analysis of phosphorylated Ser396/404 tau (PHF-1), PSD95, CRTC1, and synaptophysin in synaptosomal (B), presynaptic (C) and postsynaptic (D) fractions of WT and Tau mice. Values represent mean fold ± s.e.m. (n = 3 mice/group). Statistical analysis was determined by unpaired student’s t-test. *P < 0.05.


Human induced pluripotent stem cell (hiPSC) neural model systems can meet this demand: they provide a renewable source of material for differentiation into regional neuronal sub-types for functional assays; they can be expanded to provide a platform for screening, and they can potentially be optimized for transplantation/neurorestorative therapy. So far, however, hiPSC differentiation protocols for the generation of ventral midbrain dopaminergic neurons (mDANs) – the predominant neuronal sub-type afflicted in PD – have been somewhat restricted by poor efficiency and/or suitability for functional and/or imaging-based in vitro assays. Here, we describe a reliable, monolayer differentiation protocol for the rapid and reproducible production of high numbers of mDANs from hiPSC in a format that is amenable for autophagy/mitophagy research. We characterize these cells with respect to neuronal differentiation and macroautophagy capability and describe qualitative and quantitative assays for the study of autophagy and mitophagy in these important cells.Macroautophagy/autophagy cytoplasmic quality control pathways are required during neural development and are critical for the maintenance of functional neuronal populations in the adult brain. Robust evidence now exists that declining neuronal autophagy pathways contribute to human neurodegenerative diseases, including Parkinson disease (PD). Reliable and relevant human neuronal model systems are therefore needed to understand the biology of disease-vulnerable neural populations, to decipher the underlying causes of neurodegenerative disease, and to develop assays to test therapeutic interventions Abbreviations: AA: ascorbic acid; ATG: autophagy-related; BDNF: brain derived neurotrophic factor; CCCP: carbonyl cyanide m-chlorophenylhydrazone; dbcAMP: dibutyryl cAMP; DAN: dopaminergic neuron; DAPI: 4ʹ,6-diamidino-2-phenylindole; DAPT: N-[N--L-alanyl]-sphenylglycine; DLG4/PSD95: discs large MAGUK scaffold protein 4; DMEM: Dulbecco’s modified eagle’s medium; EB: embryoid body; ECAR: extracellular acidification rate; EGF: epidermal growth factor; FACS: fluorescence-activated cell sorting; FCCP: arbonyl cyanide p-triflouromethoxyphenylhydrazone; FGF: fibroblast growth factor; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GDNF: glia cell derived neurotrophic factor; hiPSC: human induced pluripotent stem cell; LAMP2A: lysosomal associated membrane protein 2A; LT-R: LysoTracker Red; MAP1LC3: microtubule associated protein 1 light chain 3; mDAN: midbrain dopaminergic neuron; MEF: mouse embryonic fibroblast; MT-GR: MitoTracker Green; MT-R: MitoTracker Red; NAS2: normal SNCA2; NEM: neuroprogenitor expansion media; NR4A2/NURR1: nuclear receptor subfamily group A member 2; OA: oligomycin and antimycin A; OCR: oxygen consumption rate; PD: Parkinson disease; SHH: sonic hedgehog signaling molecule; SNCA/α-synuclein: synuclein alpha; TH: tyrosine hydroxylase; VTN: vitronectin. PD affects around 1% of the population aged over 65 and around 4% aged over 85. In the UK, there are currently ~145,500 PD patients, with the expectation that by 2065 the prevalence and incidence rates for PD in the UK will have almost doubled  control processes – including transporter-mediated efflux across the plasma membrane octane ; 4ʹ,6-diamidino-2-phenylindole ; DAPT ; db-cAMP ; DMEM-high glucose ; DMEM-F12 w/o glucose ; DMEM/F-12 + Glutamax ; KnockOutTM DMEM (KO-DMEM) ; KnockOutTM serum replacement ; Essential 8 medium/E8 supplement ; F12 + Glutamax ; FGF2 ; FGF8/FGF8A ; FN1 ; galactose ; human recombinant GDNF ; Glutamax ; Hoechst 33342 ; human INS ; laminin ; LDN193189 ; LysoTracker Red ; mitomycin C ; MitoTracker Green ; MitoTracker Red ; monothioglycerol ; N2 ; neurobasal ; non-essential amino acids ; PD0325901 ; penicillin/streptomycin ; poly-L-ornithine ; polyhema ; purmorphamine ; RevitaCell ; SB431542 ; human recombinant SHH ; SYBR Green ; TGFB3 ; TF/transferrin ; VTN/vitronectin ; Y27632 .Seahorse XF base medium ; Seahorse XFp FluxPax ; Accutase ; ascorbic acid ; AZD8055 ; B27 ; bafilomycin AAntibodies used were: rabbit anti-TH ; mouse anti-TH ; anti-TUBB3 (TUJ1) ; anti-FOXA2 ; anti-LMX1A ; anti-LMX1B ; anti-TBR1 ; anti-WIPI2 ; anti-MAP1LC3B ; anti-SQSTM1 ; anti-ATP5F1B ; anti-DLG4 ; anti-S100B ; anti-SYP ; anti-SLC17A7 ; Alexa Fluor 488 anti-rabbit ; Alexa Fluor 568 anti-rabbit ; Alexa Fluor 488 anti-mouse ; Alexa Fluor 568 anti-mouse ; Alexa Fluor 647 anti-rabbit ; Alexa Fluor 647 anti-mouse ; anti-rabbit HRP ; anti-mouse HRP .2) in mitomycin c diluted in MEF media to a final concentration of 10 μg/ml.All of the functional data presented in this study was obtained using the “Normal SNCA” (NAS2) hiPSC line   was applied to 60 mm diameter tissue culture dishes for 1 h at room temperature. Culture medium (E8) was changed daily until cells reached ~80% confluency . The cells were then passaged, frozen, or differentiated. A detailed protocol for feeder-free hiPSC maintenance can be found in  in 0.1 M Na Cacodylate), washed, then dehydrated in an ethanol series before being embedded in Epon resin. Hardened resin was removed from the imaging dish, blocks were trimmed, and ~70 nm ultrathin sections cut using an Ultracut S ultramicrotome (Leica). Sections were post-stained in uranyl acetate and lead citrate and viewed using a Technai 12 120 kV transmission electron microscope (FEI). Images were obtained using a Ceta 4 k x 4 k CCD camera and processed using Fuji software.For electron microscopy, mDANs were seeded on 35 mm glass-bottomed dishes as for light microscopy (above), and treated with AZD8055 and/or BafA1 for 4 h. Cells were fixed by the addition of 2% glutaraldehyde (EM grade) for 15 min at room temperature. Following 3x washes with 0.1 M Na Cacodylate buffer, cells were osmicated (1% OsOLentiviruses were produced in HEK293 T as described : (i) PXL4/cm2. RNA was extracted by using the QIAGEN RNeasy kit following the manufacturer’s instructions. Eluted RNA was measured by a Nanodrop spectrophotometer (Thermo Fisher Scientific). cDNA was prepared using a High Capacity RNA-to-cDNA Kit  according to the manufacturer’s instructions. cDNAs were amplified using the SYBR Green Master Mix  and specific amplification primers  according to the manufacturer’s protocol, using a StepOnePlus Real-Time PCR device (Applied Biosystems). For 10 μl reactions, 2 μl of cDNA was mixed with 2 μl of RNase-free H2O, 5 μl of SYBR Green, 0.5 μl forward and 0.5 μl reverse primers. Cycle conditions set as follows: an initial denaturation at 95°C for 10 min followed 40 cycles with 95°C for 15 s (denaturation), 60°C for 30 s  and 60°C for 30 s (elongation). The relative quantification of gene expression was performed via automatic calculation of the cycle threshold (Ct) values for each target gene and was normalized for the expression of the housekeeping gene GAPDH , and was calculated using the 2(-ΔΔCt) method [Cells were plated in triplicate on 12-well plates at a density of 4x10) method .DDC: ggggaccacaacatgctgctcc/aatgcactgcctgcgtaggctg; ATG16L1: cagttacgtggcggcaggct/acaacgtgcgagccagaggg; ATG5: agcaactctggatgggattg/cactgcagaggtgtttccaa; CORIN: catatctccatcgcctcagttg/ggcaggagtccatgactgt; FOXA2: ctgggagcggtgaagatgga/acgtacgacgacatgttcatggag; GAPDH: ttgaggtcaatgaaggggtc/gaaggtgaaggtcggagtca; KCNJ6 (GIRK2): atggatcaggacgtcgaaag/atctgtgatgacccggtagc; LAMP2: cgttctggtctgcctagtcc/cagtgccatggtctgaaatg; LMX1A: agagctcgcctaccaggtc/gaaggaggccgaggtgtc; NANOG: ttgggactggtggaagaatc/gatttgtgggcctgaagaaa; NR4A2: gtgttcaggcgcagtatgg/tggcagtaatttcagtgttgg; POU5F1 (OCT4): gacaacaatgagaaccttcaggaga/ctggcgccggttacagaacca; OPTN: tgctgagtccgcacataga/gggtccatttcctgtgctt; SQSTM1: ctgggactgagaaggctcac/gcagctgatggtttggaaat; PINK1: gcctcatcgaggaaaacagg/gtctcgtgtccaacgggtc; TH: gccgtgctaaacctgctctt/gtctcaaacaccttcacagctc.The following primers were used (Gene: 5ʹ-3ʹ forward/5ʹ-3ʹ reverse): 4 cells/cm2 and allowed to terminally differentiate for 10 d. 3 d after plating, cells were transduced with PRRL human SYN1-GFP lentiviruses. 10 d post-plating, cells were treated for 3 h with DMSO (vehicle control) or 10 μM CCCP and were then washed once with PBS and probed with 50 nM MT-R for 30 min (37°C/5% CO2). Following 2 washes with PBS, MT-R labeled cells were dislodged into single cells by 10 min incubation in Accutase (37°C/5%CO2), centrifuged (100 x g for 1 min) and resuspended in fresh pre-warmed DMEM. Flow cytometry was performed using a BD Influx cell sorter (BD Biosciences). Briefly, over 50,000 viable cells were recorded using the DRAQ7 DROP & GO viability dye . Cells were excited using 488 nm, 552 nm and 640 nm lasers, and fluorescence emissions were captured for GFP (488 nm 530/40 nm BP), MT-R (552 nm 610/20 nm BP) and DRAQ7 (640 nm 750 LP) respectively. Data were analyzed using FlowJo v10.3 software (Treestar).50-55-d-old differentiated neuronal progenitors were plated in 12-well plates at a density of 6 × 104 cells/cm2 and allowed to differentiate in N2B27 media supplemented with BDNF, GDNF, AA, db-cAMP and DAPT. Depending on the experimental conditions, 1 or 3 d prior to the assay, media was replaced with Seahorse bioenergetics media consisting of 1:1 DMEM-F12 (no glucose) and Neurobasal (no glucose) supplemented with 20 ng/ml BDNF, 20 ng/ml GDNF, 200 μM AA, 500 μM db-cAMP, 10 μM DAPT, 0.25 mM sodium pyruvate and 10 mM galactose or 10 mM glucose . On the day of the assay, culture media was replaced with Seahorse XF base medium supplemented with 1 mM sodium pyruvate, 2 mM glutamine, and 10 mM glucose or 10 mM galactose  and the cells were incubated at 37°C (without CO2) for 1 h before the assay. Data was extracted and analyzed automatically by the Seahorse Wave software.To measure oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) in the presence of glucose or galactose, an Agilent Seahorse XFp Analyzer  with cell energy phenotype test kit was used (according to the manufacturer’s instructions). 54-d-old neural progenitors were plated on 8-well Seahorse XFp Cell culture mini-plates  at a density of 4 × 10Mean differences were analyzed by student’s t-test, one-way and two-way ANOVA  followed by Tukey’s  or Danette’s  multiple comparison posthoc tests. Statistical significance was determined as *p < 0.05, **p < 0.01 and ***p < 0.001 using GraphPad Prism 6 software .Click here for additional data file.


II coordination polymer constructed from 4,4′-bipyridine  and m-chlorobenzoate (3-Clbenz), {[Co34(3-Clbenz)6(H2O)2]·2CH3OH}n, are reported.The synthesis, crystal structure and properties of a novel ladder-chain Co 3(C7H4ClO2)6(C10H8N2)4(H2O)2]·2CH3OH}n, was synthesized and characterized. The structure contains two CoII centres with different octahedral environments, [Co(1)N3O3] and [Co(2)N2O4]. The O-donating 3-chlorobenzoate anions (3-Clbenz) act as the terminal ligands, while the N-donating 4,4′-bipy molecules play the role of linkers. The Co(1) ions are linked by 4,4′-bipy molecules into linear chains. Two such chains are joined by [Co(2)(3-Clbenz)2(H2O)2] units via two 4,4′-bipy bridging ligands, thus forming the ladder-chain structure. The crystal packing of the title compound is stabilized by supramolecular interactions, such as hydrogen bonding, π–π and halogen⋯π contacts, giving a three-dimensional framework. The spectroscopic and thermal properties of the title compound have also been investigated.A novel ladder-chain cobalt(II) coordination polymer, {[Co N- and O-donating ligands such as 4,4′-bipyridine  and benzoate derivatives have been intensively developed , which is expected to support crystal structures via π–π and halogen⋯π intermolecular interactions, together with the 4,4′-bipy organic linker and have synthesized the new CoII coordination polymer {[Co34(3-Clbenz)6(H2O)2]·2CH3OH}n, which has an interesting one-dimensional ladder-chain structure. This report describes the synthesis, crystal structure, spectroscopic and thermal properties of the title compound.The exploration and synthesis of new one-dimensional coordination polymers based on transition metals and mixed 2+ ions, three 3-Clbenz anions, two 4,4′-bipy molecules, one coordinated water molecule and one methanol solvate molecule as shown in Fig. 12+ ions (containing Co2), is situated at an inversion centre. One pyridine ring (C1–C5) and the methanol solvate molecule are disordered over two sets of sites with occupancies of 0.584 (19):0.416 (19) and 0.72 (3):0.28 (3), respectively. Both Co2+ ions are six-coordinated and have octahedral environments. The Co1 ion is coordinated by three nitrogen atoms from three 4,4′-bipy bridging ligands and three oxygen atoms from the carboxylate groups of one monodentate and one bidentate 3-Clbenz ligands, providing a distorted octahedral geometry with angles O2—Co1—O1, O2—Co1—O3 and O2—Co1—N1 of 59.88 (6), 119.93 (7) and 148.87 (7)°, respectively. The Co2 ion is coordinated by two nitrogen atoms from two 4,4′-bipy bridging ligands and four oxygen atoms from two monodentate 3-Clbenz ligands and two coordinated water molecules. The angles in its environment deviate from ideal values no more than by 3.5°. There is an intramolecular hydrogen bond in the coordination environment of Co2 between the aqua and 3-Clbenz ligands . Aromatic π–π interactions involving the phenyl rings of two 3-Clbenz ligands have an intercentroid Cg6⋯Cg7  separation of 3.917 (2) Å . There are also halogen⋯π interactions between the 3-Clbenz ligands and the pyridine rings of 4,4′-bipy ligands with C24—Cl1⋯Cg5  = 3.5833 (14) Å and C31—Cl2⋯Cg4  = 3.7558 (15) Å . These interactions stabilize the structure, leading to a three-dimensional supramolecular framework , aromatic π–π and Cl⋯π interactions  has a characteristic broad peak centred at 3330 cm−1 assigned to the O—H stretching vibrations of coordinated water molecules and the methanol solvate. The strong and sharp peaks at about 1608 and 1382 cm−1 are attributed to the asymmetric and symmetric COO− stretching vibration of the monodentate 3-Clbenz ligands, and the peaks appearing at about 1557 and 1488 cm−1 are attributed to the asymmetric and symmetric COO− stretching vibration of the chelating 3-Clbenz ligand , suggesting coordinated 4,4′-bipy ligands  of the title compound shows d–d transitions with two broad bands at 489 and 1099 nm, assigned to the ν3: 4Tg1 → 4Tg1(P) and ν1: 4T1g → 4Tg2 transitions, respectively  of the title compound used to check the phase purity show good accordance with its simulated PXRD pattern generated from the single-crystal X-ray diffraction data, confirming that the title compound has high phase purity. The TGA curve  shows the thermal stability of the title compound below 325°C. The first complex step with a weight loss of 29.57%  was found in the temperature range from 100 to 325°C, which was attributed to the loss of methanol molecule of crystallization, two coordinated water and three 3-Clbenz molecules. Then, the structure starts to collapse with a weight loss of 49.24%  in the temperature range from 325–685°C that can be attributed to the removal of three remaining 3-Clbenz and three remaining 4,4′-bipy molecules. After that, the residual product is assumed to be CoO.3(dca)2(nic)4(H2O)8]·2H2O 2(nic)4(H2O)8]·2H2O 2·6H2O  in 10 mL of MeOH/H2O (v:v = 8:2) solution. After stirring for 30 min, a methanolic solution (5 mL) of m-chlorobenzoic acid  was added slowly, and the mixture was stirred continuously at room temperature for 15 minutes. The resulting clear red solution was allowed to evaporate slowly in air. After 4 days, red rod-shaped crystals suitable for single-crystal X-ray diffraction were obtained. Yield 115.2 mg . Analysis calculated for C84H68Cl6Co3N8O16: C, 54.98; H, 3.74; N, 6.11%. Found: C, 53.28; H, 3.60; N, 6.50%. IR : 3330(w), 2348(w), 1608(s), 1557(s), 1488(w), 1415(s), 1382(s), 1263(w), 1219(m), 1145(w), 1068(m), 1031(w), 1010(w), 817(m), 808(m), 760(m), 731(m), 674(w), 657(w), 631(m), 574(w), 499(w), 439(w).A methanolic solution (5 ml) of 4,4′-bipy  was added to a solution of Co(NOUiso(H) = 1.5 Ueq(C)], C—H = 0.93 Å for aromatic [Uiso(H) = 1.2 Ueq(C)]. The oxygen-bound hydrogen atom of methanol was positioned with O—H = 0.82 Å [Uiso(H) = 1.5Ueq(O)], and the OH group was allowed to rotate (AFIX 147). Hydrogen atoms of the coordinated water molecule were located in the differential electron density map and refined with the O—H distance contrained to 0.84 Å.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989022000731/yk2163sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989022000731/yk2163Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989022000731/yk2163sup4.pdfSupporting data of crystal structure, characterization and physical properties. DOI: 2143539CCDC reference:  crystallographicinformation; 3D view; checkCIF reportAdditional supporting information: