The high-Z atoms of Lu and Cs can deposit more X-rays for generating enhanced hydroxyl radicals (·OH). Meanwhile, the LA ligand will guide CsLu2F7-LA to target monocarboxylic acid transporter (MCT) and impede the transportation of free LA, leading to decreased glycolysis and DNA damage repair. Consequently, the curative effect of RT will be enhanced and the strategy of LA accumulation induced radiosensitization is proved by in vivo and in vitro experiments, which will bring prospects for enhanced RT with nanomedicine.Deficient deposition of X-rays and strong capacity of repairing damage DNA of cancer cells limit the effect of radiation therapy (RT). Herein, we synthesize CsLuThe online version contains supplementary material available at 10.1186/s12951-022-01694-1. Generally, ·OH will attack DNA molecules, induce irreversible DNA double-strand breaks (DSBs), and finally lead to the proliferative death of cancer cells [Radiation therapy (RT) is widely used in clinic for the treatments of cancer –3. Accorer cells , 6. Unfoer cells , 8. Secoer cells –12. For er cells , 13. HenRecently, the progress of nanomedicine brings more chances for radiosensitization –21. On t2F7 nanoparticles with LA ligands (CsLu2F7-LA) for enhancing the effect of RT. As shown in Fig. 2F7 (CsLu2F7-OA), LA can easily replace OA to obtain water-soluble CsLu2F7-LA because the strong acid can replace the weak one [2F7-LA can deposit more X-rays in tumor area  is the final product in the process of glycolysis and plays an important role in tumor progression . Cancer weak one , 41. As 3, cesium acetate (CsAc), lactic acid (LA), oleic acid (OA), 1-octadecene (ODE) and Rhodamine B (RhB) were purchased from Macklin. NH4F, methanol, ethanol and cyclohexane were purchased from Sinopharm Chemical Reagent Co., Ltd. Phosphate buffered solution (PBS), dulbecco’s modified eagle medium (DMEM) and fetal bovine serum (FBS) were obtained from Adamas Life. Cell counting kit-8 (CCK-8), histone H2AX rabbit polyclonal antibody, fluorescein isothiocyanate (FITC), FITC-labeled goat anti-rabbit IgG (H + L), DAPI staining kit, Ki67 staining kit, 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) assay kit, 2,7-Dichlorodi-hydrofluorescein diacetate (DCFH-DA) assay kit, ATP assay kit, hematoxylin and eosin (H&E) staining kit, TUNEL apoptosis assay kit, calcein/PI cell viability/cytotoxicity assay kit, Annexin V-FITC/PI apoptosis assay kit were bought from Beyotime.LuClTransmission electron microscope (TEM) image was carried out by FEI Talos F200X. X-ray diffraction (XRD) was measured by Rigaku D/MAX-2250V. Dynamic light scattering was measured by Brookhaven omni. X-ray photoelectron spectroscopy (XPS) spectra was measured by Thermo Fisher Scientific ESCALAB 250XI. Confocal laser scanning microscopy was carried out on Leica TCS SP8 STED 3X. Fourier transform infrared spectroscopy (FTIR) was measured by Bruker Thermo Fisher Nicolet 6700. The concentration of Lu element was measured by inductively coupled plasma optical emission spectrometer . The fluorescence microplate system was TECAN SPARK. Flow Cytometer was BD FACSCalibur. Radiation therapy was carried out by clinical 220 keV X-ray .3, 20 mL OA and 10 mL ODE were mixed in a flask with three necks and heated to 150 ℃ under the protection of N2 for 1 h, and then cooled to room temperature. 7 mmol NH4F and 1 mmol CsAc were poured into the flask and stirred for 1 h. Then this mixture was heated to 290 °C for 1.5 h and cooled to room temperature subsequently. After washed with ethanol and cyclohexane for three times, the obtained CsLu2F7-OA were dispersed in 10 ml cyclohexane. 10 ml as-prepared CsLu2F7-OA solution in cyclohexane and 10 ml LA solution in water (0.1 M) were mixed and stirred for 4 h. Then this mixture was washed with ethanol for three times, the obtained CsLu2F7-LA were dispersed in 10 ml water.2 mmol LuCl2F7-LA (500 ppm) or water (control group) were placed in centrifuge tubes. These tubes were irradiated with 0 Gy, 5 Gy, 10 Gy, 15 Gy and 20 Gy of X-rays. Supernatants were collected after centrifugation. The ·OH yield was represented by the degradation rate of RhB (absorbance at 554 nm).4 ml solution contained RhB (5 ppm) with CsLu143B cells (human osteosarcoma cells) and HUEVC  cells were purchased from Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. Kunming mice and Balb/c nude mice were purchased from Shanghai SLAC Laboratory Animal Co. Ltd. All the experiments in vivo were approved by the animal ethics committee of Shanghai Jiao Tong University, and the ethics number was 20210918-02.2F7-LA at different concentrations and cultured for another 24 h. 100 μl culture media containing 10 μl CCK-8 solution was added and co-incubated with cells for 2 h. The absorbance was measured using a microplate reader at 450 nm.143B cells or HUVEC cells  were seeded in the 96-well plates and cultured at 37 °C for 24 h. Next, the medium was replaced by the fresh DMEM medium containing LA and CsLu2F7-LA (50 ppm) respectively for another 24 h. The cells were irradiated with X-ray (0 Gy or 4 Gy). After 12 h, flow cytometer with Annexin V-FITC/PI double staining was used to evaluate cell apoptosis.143B cells  were seeded in 6-well plates and cultured for 24 h. Then the cells were incubated with DMEM medium and CsLu2F7-LA (50 ppm) respectively for another 24 h. The cells were irradiated with X-ray (0 Gy or 4 Gy). After 1 h, the cells were fixed by 4% paraformaldehyde for 15 min and washed three times with PBS. 0.2% Triton X-100 was added to penetrate cells for 10 min. Next, the 1% BSA dissolved in PBS was used to block the cells for another 1 h at room temperature. After incubation with γ-H2AX antibody overnight at 4 °C, the cells were treated with Anti-Rabbit IgG (H + L), F(ab')2 Fragment (Alexa Fluor®488 Conjugate) for 1 h and then stained with DAPI for 15 min. Lastly, the fluorescence of γ-H2AX was observed by a confocal fluorescence microscope.143B cells  were seeded in confocal dishes (diameter: 20 mm) and cultured for 24 h. Then the cells were incubated with DMEM medium and CsLu2F7-LA (50 ppm) respectively for another 24 h. The cells were irradiated with X-ray (4 Gy). After 24 h, the cells were incubated with calcein-AM and PI for 0.5 h. For ROS detection, the cells were incubated with DCFH-DA for 0.5 h. After incubation, the fluorescence was observed by a confocal fluorescence microscope.143B cells  were seeded in confocal dishes (diameter: 20 mm) and cultured for 24 h. Then the cells were incubated with DMEM medium and CsLu2F7-LA (50 ppm) respectively for another 24 h. After 24 h, the cells were incubated with BCECF for 0.5 h. After incubation, the fluorescence was observed by a confocal fluorescence microscope.143B cells  were seeded in confocal dishes (diameter: 20 mm) and cultured for 24 h. Then the cells were incubated with DMEM medium and CsLu2F7-LA (50 ppm) for another 24 h and subsequently irradiated with 0 Gy, 2 Gy, 4 Gy, 6 Gy X-rays. After being cultured with fresh medium for 10 days, the cells were fixed with absolute methyl alcohol and stained with hematoxylin and eosin. The number of colonies was counted by Image-J.143B cells were seeded in the 6-well plates at various densities of 500, 500, 1000, and 2000 cells/well for 24 h. Then the cells were incubated with DMEM medium and CsLu2F7-LA. The Kunming mice  were injected with CsLu2F7-LA (0–80 mg/kg) through the tail vein. The main tissues  of Kunming mice were dissected for H&E staining at 30 days post-injection. The blood of Kunming mice was harvested for blood routine test and biochemical examination.The standard H&E staining and blood parameter were conducted to monitor the biocompatibility of CsLu6 cells) were injected subcutaneously into Balb/c nude mice . When the tumor volume reached about 100 cm3, the mice were divided four groups randomly: (i) Control, (ii) CsLu2F7-LA, (iii) Control + X-ray, (iv) CsLu2F7-LA + X-ray. PBS (10 μL) and CsLu2F7-LA  were injected into tumors directly. After 24 h, the tumors of groups iii and iv were irradiated with X-rays (6 Gy). After 48 h, the H&E, TUNEL and Ki67 staining of the tumor tissues were performed by commercially available kits. The body weight and tumor volume of the mice were measured every 2 days.To set up the xenograft tumor model, 143B cells  to react at 150 ℃ to obtain the Lu precursor (lutecium oleate). Then this precursor is mixed with cesium acetate (CsAc) and ammonium fluoride (NH4F), and react at 290 ℃ to obtain CsLu2F7-OA. To obtain CsLu2F7-LA, the CsLu2F7-OA cyclohexane solution is mixed with LA water solution for 24 h, during this process LA will gradually replace OA. As shown in Fig. 2F7-OA is monodispersed with a size of ~ 10 nm, while the hydrodynamic radius of CsLu2F7-OA  and X-ray photoelectron spectroscopy (XPS) also show that CsLu2F7-LA contain Cs, Lu and F elements . As shown in Additional file 2F7-LA can induce more ·OH than control group (DI water), presenting that CsLu2F7-LA can deposit more X-rays.As shown in Fig. yrolysis , 43. We 2F7-LA, we clarify if they could disturb the glycolysis through blocking MCT. As shown in Fig. 2F7-LA present little cytotoxicity to human umbilical vein endothelial cell (HUVEC) even at the concentration of 100 ppm (Lu atoms). While as shown in Fig. 2F7-LA makes obvious damage to 143B cells (human osteosarcoma cell). LA is a kind of molecule that exists in the human body, and present little cytotoxicity to 143B cells -5-(and-6)-carboxyfluorescein (BCECF) staining. If CsLu2F7-LA block MCT, LA will accumulate in cells and the pH will decrease. As shown in Fig. 2F7-LA group is lower than that of control group, indicating CsLu2F7-LA can decrease the pH of 143B cells. Additionally, as shown in Fig. 2F7-LA group further prove that the accumulation of LA will impede the process of glycolysis. The cytophagy of CsLu2F7-LA is detected by confocal microscopy. CsLu2F7-LA are decorated with fluorescein isothiocyanate (FITC) through electrostatic adsorption. After co-culture for 4 h, the fluorescence of FITC is found in 143B cells, indicating that CsLu2F7-LA can enter into cells , aspartate transaminase (AST), alkaline phosphatase (ALP), urea and creatinine (CRE) of different dosage of CsLu2F7-LA also exhibit few differences  staining, Ki67 staining, and terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) staining of tumor sections also show that the combination of CsLu2F7-LA and X-ray can induce the most apoptosis and necrosis . Figure S2. FTIR spectra of CsLu2F7-OA and CsLu2F7-LA. Figure S3. Hydrodynamic size of CsLu2F7-LA dispersed in DI water, saline and DMEM at 1st day, 7th day and 14th day. Figure S4. Cs 3d, Lu 4d and F 1s X-ray photoelectron spectroscopy (XPS) spectrum. Figure S5. The yield of ·OH in solutions upon irradiation of 0 Gy, 5 Gy, 10 Gy, 15 Gy and 20 Gy X-rays . Four asterisks indicate P < 0.0001 according to Student’s two-tailed t-test. Figure S6. Relative release of Lu atoms of CsLu2F7-LA in saline . Figure S7. Cytophagy of CsLu2F7-LA (50 ppm). CsLu2F7-LA were decorated with FITC. Scale bar: 100 μm. Figure S8. Apoptotic analysis measured by flow cytometry. Figure S9. Relative intensity of γ-H2AX in each group . Figure S10. Biological compatibility evaluation of CsLu2F7-LA. Figure S11. H&E staining of major organs  30 days post intravenous injection of different dosage of CsLu2F7-LA. Scale bar: 100 μm.


The Sgalactose—Cthiophene bond is short. The molecules are connected by two ‘weak’ hydrogen bonds and a short N⋯S contact.The benzothiazole and thiophene rings are almost coplanar. The NH 32H29BrN2O10S3, the benzothiazole and thiophene ring systems subtend an interplanar angle of 7.43 (12)°. The NH2 group forms intramolecular hydrogen bonds to Nthiazole and Ocarbonyl. The Sgalactose—Cthiophene bond is short [1.759 (2) Å]. The molecules are connected to form ribbons parallel to the b axis by two ‘weak’ hydrogen bonds and a short Namino⋯Sgalactose contact.In the title compound, C The 1H NMR spectrum of compound 3 showed the anomeric proton as a doublet at δ = 5.39 p.p.m. with a spin–spin coupling constant  confirming the β-configuration. The other six protons of galactose resonated at δ 4.00–5.30 p.p.m. In order to establish the structure of the product unambiguously, its crystal structure was determined and is reported here. To the best of our knowledge, this is the first reported X-ray structure of the new compound type benzothiazole-2-thiophene thioglycoside.As a part of our current plan directed toward discovering synthetic methodologies for the preparation of ld Fig. 1. It has 3 is shown in Fig. 22 group to the thiazole nitrogen atom and the C=O group  and 1.759 (2) Å, respectively; the latter is significantly shorter than the values found for similar compounds in search of the Cambridge Structural Database  for purely organic galactose derivatives substituted with a sulfur atom at the anomeric carbon. There were 22 hits, of which two were axially substituted  at the anomeric carbon of the sugar (here C31) is confirmed, as is the axial configuration of the substituent at C34, characteristic of galactose. The galactose ring displays a slightly flattened chair conformation, with absolute torsion angles < 50° about C32—C33 and C33—C34. The configurations at C31–C35 are x, 1 + y, z). The net effect is to form ribbons of molecules parallel to the b axis  was dissolved in acetone (10 ml) containing 0.5 ml of aq. KOH . The mixture was warmed to 50°C for 15 min. After cooling, a solution of 2,3,4,6-tetra-O-acetyl-β-d-galactopyranosyl bromide 2  in acetone (10 ml) was added dropwise over 30 min. The reaction mixture was stirred at room temperature and monitored by TLC until the reaction was complete (8 h). The residue was washed with distilled water to remove KBr, then dried and crystallized from ethanol to produce compound 3 : ν 3406–3281 (NH2), 2923 (ArCH), 1748 (4Ac-CO), 1720 (CO); 1H NMR : δ 1.89, 1.91, 1.94, 2.01 , 4.00–4.02 , 4.32 , 5.15 , 5.25–5.30 , 5.39 , 7.52 , 7.61 , 7.77–7.79 , 8.14 , 8.21 , 8.93 ; Analysis: calculated for C32H29 BrN2O10S3 (777.68): C, 49.42; H, 3.76; N, 3.60; S, 12.37%. Found: C, 49.39; H, 3.73; N, 3.67; S, 12.40%.Yellow solid, yield 65%, m.p. 403–405 K (EtOH); IR  I, global. DOI: 10.1107/S2414314622004126/mw2186Isup2.hklStructure factors: contains datablock(s) I. DOI: 2167334CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Both molecules (I and II) have chiral centers at the carbon atoms where the triazine rings of molecules I and II are attached to the phenyl ring. In the crystal, molecules I and II are linked by intermolecular N—H⋯N, N—H⋯O and C—H⋯N hydrogen bonds through the solvent dimethylformamide molecule into layers parallel to (001). In addition, C—H⋯π interactions also connect adjacent molecules into layers parallel to (001). The stability of the molecular packing is ensured by van der Waals interactions between the layers. The Hirshfeld surface analysis indicates that N⋯H/H⋯N , H⋯H  and C⋯H/H⋯C  interactions are the most significant contributors to the crystal packing.The title compound, 2C The triazine ring (N1–N3/C1–C3) in I adopts a distorted envelope conformation with puckering parameters  = 0.2149 (17) Å and φ(2) = 246.1 (4)°, while the triazine ring (N7–N9/C13–C15) in II has an envelope conformation . Fig. 2I and II in the asymmetric unit, with an r.m.s. deviation of 0.170 Å. The phenyl ring of molecule I is disordered over two sets of sites with an occupancy ratio of 0.67 (3):0.33 (3) (major component C4–C9 and minor component C4A–C9A). These disordered phenyl rings are at a dihedral angle of 6.0 (13)° to each other and the major and minor disorder components make dihedral angles of 86.9 (5) and 87.5 (12)°, respectively, with the mean plane of the triazine ring of molecule I. The phenyl ring (C16–C21) in II makes a dihedral angle of 86.65 (9)° with the mean plane of the triazine ring. There is one stereogenic center in both racemic molecules and the chirality about atoms C1 in I and C13 in II is S in the chosen asymmetric unit. Molecules I and II have normal geometric parameters.The title compound Fig. 1 contains3.I and II are linked by intermolecular N—H⋯N, N—H⋯O and C—H⋯N hydrogen bonds .Hirshfeld surfaces for both molecules were calculated using b; 38.3% contribution for I; 35.0% for II) are the major factor in the crystal packing with H⋯H  and C⋯H/H⋯C  interactions representing the next highest contributions. The percentage contributions of comparatively weaker interactions are N⋯C/C⋯N , N⋯N , O⋯H/H⋯O , C⋯C  and O⋯N/N⋯O . The data comparison shows that the surroundings of molecules I and II are quite similar. Short contacts are summarized in Table 2Fig. 84.viz. 3-(p-chlorophenyl)-4-[ methyl]-4,6-diphenyl-2-oxo-1,2,3,4-tetrahydro-13,5-triazine guanidinium chloride , benzaldehyde  and malononitrile  was refluxed in methanol for 5 h. The solvent was subsequently removed in vacuo, and the residue was recrystallized from methanol using charcoal. Crystals suitable for X-ray analysis were obtained by slow evaporation of a DMF solution. Colorless solid (47%); Analysis calculated for C27H27N13O (M = 549.6): C 59.01, H 4.95, N 33.13; found: C 58.98, H 4.89, N 33.07%. 1H NMR (DMSO-d6) δ 8.58 (NH), 8.30 (NH), 7.95 (CH), 7.31–7.45 , 5.62 (CH), 2.72 and 2.88 (2CH3). 13C NMR (DMSO-d6) δ 165.17, 162.44, 155.68, 141.91, 128.78, 128.72, 125.73, 119.45, 119.19, 61.80, 37.67, 35.87. ESI–MS: m/z: 550.5 [M + H]+.A mixture of (6.Uiso(H) = 1.2 or 1.5Ueq(C)] and were included in the refinement in the riding-model approximation. The N-bound H atoms were located in a difference-Fourier map and were fixed at their found positions and refined with a riding model with Uiso(H) set to 1.2Ueq(N). In molecule I, the C6(C6A)–C9(C9A) atoms in the C4–C9 phenyl ring are disordered over two sets of sites with an occupancy ratio of 0.67 (3):0.33 (3). The H atoms of a methyl group (C26) of the dimethylformamide solvent were refined as disordered [C—H = 0.98 Å and Uiso(H) = 1.5Ueq(C)], using AFIX 127 (rotating disordered methyl group) and a free variable for the two groups of H atoms with an occupancy ratio of 0.66 (3):0.34 (3). For the two disordered parts of the phenyl ring of molecule I, the corresponding ring of molecule II, which is not disordered, was used as a template using a SAME command. Furthermore, the displacement parameters of the C atoms of the major and minor components of the disordered phenyl ring were restrained with a SIMU 0.02 command, while the displacement parameters of the C atoms of the major and minor components of the disordered phenyl ring attached to the triazine ring of molecule I (C4 and C4A) were constrained to have identical ADPs using an EADP instruction. All the C—C bonds between the phenyl and triazine rings were restrained to be similar to each other using a SADI instruction.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989022006910/zl5032sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989022006910/zl5032Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989022006910/zl5032Isup3.cmlSupporting information file. DOI: 2184452CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Macro-autophagy/Autophagy is an evolutionarily well-conserved recycling process to maintain the balance through precise spatiotemporal regulation. However, the regulatory mechanisms of biomolecular condensates by the key adaptor protein p62 via liquid-liquid phase separation (LLPS) remain obscure.In this study, we showed that E3 ligase Smurf1 enhanced Nrf2 activation and promoted autophagy by increasing p62 phase separation capability. Specifically, the Smurf1/p62 interaction improved the formation and material exchange of liquid droplets compared with p62 single puncta. Additionally, Smurf1 promoted the competitive binding of p62 with Keap1 to increase Nrf2 nuclear translocation in p62 Ser349 phosphorylation-dependent manner. Mechanistically, overexpressed Smurf1 increased the activation of mTORC1 (mechanistic target of rapamycin complex 1), in turn leading to p62 Ser349 phosphorylation. Nrf2 activation increased the mRNA levels of Smurf1, p62, and NBR1, further promoting the droplet liquidity to enhance oxidative stress response. Importantly, we showed that Smurf1 maintained cellular homeostasis by promoting cargo degradation through the p62/LC3 autophagic pathway.These findings revealed the complex interconnected role among Smurf1, p62/Nrf2/NBR1, and p62/LC3 axis in determining Nrf2 activation and subsequent clearance of condensates through LLPS mechanism.The online version contains supplementary material available at 10.1186/s13578-023-00978-9. The biological macromolecules, such as proteins and nucleic acids, spontaneously transition to liquid droplets  to maintain homeostasis by controlling nuclear function, regulating cellular quality control, and organizing biochemical networks . They diHO1 (Heme Oxygenase 1), NQO1(NAD(P)H Quinone Dehydrogenase 1), and p62 [The ubiquitin-proteasome system (UPS) and autophagy are the main cellular clearing systems to maintain protein homeostasis in response to extreme environmental stress  . The p62 and p62 . Especia and p62 , 24. HowOncogenic E3 ligase SMAD Specific E3 Ubiquitin Protein Ligase 1 (Smurf1) is highly expressed in cancer cells and recognized as a new mediator of selective autophagy by interacting with the nascent autophagosome membrane –27. ThisKO (Smurf1−/−) and Wild-type (Smurf1+/+) cells with a proteasome inhibitor MG132. The mouse embryonic fibroblasts (MEFs) were generated from Smurf1−/− and Smurf1/++ mice. We found more RFP-p62 puncta in Smurf1/++ MEFs than Smurf1−/− MEFs, under MG132 treatment  in LN229 cells. The number and size of p62 puncta failed to respond to Flag-Smurf1C699A under MG132 treatment compared with Flag-Smurf1, suggesting Smurf1 promoted the formation of p62 puncta in its E3 ligase activity-dependent manner  of RFP-p62 in GFP-Smurf1+/RFP-p62+ puncta were generally at 2 ± 1 min compared to GFP-p62 single puncta at 12 ± 3 min, indicating that Smurf1 facilitate p62 to induce exchanging molecules with the surrounding environment  was shorter than GFP-Smurf1 single puncta (3 ± 1 min)  puncta, suggesting that Smurf1-induced exchanging molecules may depend on p62 phase separation ability  assay . To deteSmurf1−/−) mice compared to Smurf1/++ MEFs  in p62WT and p62K7A/D69A group but not in p62S349A and control group , thus promoting activation of Nrf2 . S403 ph+p62+ puncta was increased in Smurf1 overexpression group and decreased in Smurf1 or p62 knockdown group under MG132 treatment   and RFP+GFP− signal (in autolysosomes) were both dramatically increased in the cells with overexpressed Smurf1 compared to the control cells, while only a small amount RFP+GFP+ signal was exhibited in the knockdown of Smurf1, further indicating that Smurf1 induces the autophagic formation and degradation of p62  and increased number of RFP+GFP− sign (in autolysosomes) compared with si-Control in Flag-Smurf1 LN229 cells  in si-Control group compared to si-Smurf1 group in LN229 , and NBR1 knockdown (2 ± 1 min) decreased the min, andNext, we knocked down NBR1 or p62 in LN229 cells with Smurf1 overexpression or Smurf1 knockdown. We found that the Smurf1-mediated increase of NBR1 depended on p62 protein level, evidenced by the p62 knockdown diminished the response of NBR1 to overexpression or knockdown of Smurf1  production by recruiting Keap1 and subsequently activating the transcription factor Nrf2 , 33, 34,ner Fig. A. The qRner Fig. B. These ion Fig. C. To fur229 Fig. D. The qRQO1 Fig. E. Smurf1ess Fig. E. Furtheent Fig. F.Fig. 7N2O2 and Flag-Smurf1-induced p62 -inducible protein. It collaborated with p62 and NBR1 during the activation of the Keap1-Nrf2 oxidative stress response pathway, promoting the formation and degradation of p62-liquid droplets and Nrf2 full-activation , thereby increasing p62 liquid droplet formation. Most importantly, Smurf1 is also upregulated by Nrf2 in oxidative stress  and human glioblastoma cell lines LN229 were purchased from the American Type Culture Collection (ATCC) and reauthenticated by Short Tandem Repeat  Corporation Limited). Cells were grown in DMEM  media supplemented with 10% fetal bovine serum , and penicillin-streptomycin , a humidified atmosphere of 5% COCells were transfected with plasmids or siRNA using Lipofectamine 2000 reagent (Invitrogen) or Lipofectamine® RNAiMax Reagent (Invitrogen) following the supplier’s instructions. The cells were collected and analyzed by western blot.2O2 .Cells were treated with dimethyl sulfoxide , Z-Leu-Leu-Leu-al , EBSS , Bafilomycin , Rapamycin  or HThe following human siRNAs were used: si-Control was purchased from JTS scientific; si-Smurf1: 5ʹ-GCGUUUGGAUCUAUGCAAATT-3ʹ; si-p62: 5ʹ-GGCUGAAGGAAGCUGCCUU-3ʹ; si-Nrf2: 5ʹ-GAAUGGUCCUAAAACACCATT-3ʹ; si-NBR1: 5ʹ-AAACCUGACUUUGGCUUCCACAGAA-3ʹ.Total RNA was extracted from each sample using TRNzol Universal reagent  according to the manufacturer’s instructions. 500 ng of RNA was used as a template to generate cDNA using the ABScript II RT Mix for qRT-PCR with gDNA Remover  according to the manufacturer’s instructions. For PCR amplification of cDNA, 5 μl of the reverse transcription reaction solution was used with sense and anti-sense primers of the gene of interest using 2 × Universal SYBR Green Fast qRT-PCR Mix , and primers corresponding to β-actin were included as an internal control. Primer sequences were NBR1, 5ʹ-AGGAGCAAAACGACTAGCTGC-3ʹ(forward) and 5ʹ-TCTGGGGTCTTCATGTCTGAT-3ʹ(reverse); p62, 5ʹ-GACTACGACTTGTGTAGCGTC-3ʹ(forward) and 5ʹ-AGTGTCCGTGTTTCACCTTCC-3’(reverse); β-actin, 5ʹ-CATGTACGTTGCTATCCAGGC-3ʹ(forward) and 5ʹ-CTCCTTAATGTCACGCACGAT-3ʹ(reverse); Smurf1, 5ʹ-AGATCCGTCTGACAGTGTTATGT-3’(forward) and 5ʹ-CCCATCCACGACAATCTTTGC-3ʹ(reverse); NQO1, 5ʹ-GAAGAGCACTGATCGTACTGGC-3ʹ(forward) and 5ʹ-GGATACTGAAAGTTCGCAGGG-3’(reverse); HO1, 5ʹ-AAGACTGCGTTCCTGCTCAAC-3ʹ(forward) and 5ʹ-AAAGCCCTACAGCAACTGTCG-3ʹ(reverse).Western blot was used to analyze protein expression as described. In brief, cells were collected and lysed with RIPA buffer  supplemented with 1 mM PMSF and 1% phosphatase inhibitor (BOSTER). Protein lysates with 5 × loading buffer  were sonicated and boiled for 10 min. Protein samples were first resolved by SDS-PAGE, then transferred to a nitrocellulose membrane, and subsequently incubated with the primary antibody. After incubation with peroxidase-conjugated secondary antibodies, the signals were visualized by enhanced chemiluminescence according to the manufacturer’s instructions. The following primary antibodies were used: p62 , p-p62 Ser403 , p-p62 Ser349 , β-actin , Nrf2 , NDP52 , NBR1 , LC3B , OPTN , Smurf1 , GFP , HA , Flag , c-Myc , Keap1 , BAG3 , p-mTOR (Ser2448) , mTOR , p-p70S6K (Thr389) , p70S6K . The following secondary antibodies were used: Horseradish Peroxidase-conjugated goat anti-rabbit IgG , Horseradish Peroxidase-conjugated goat anti-mouse IgG .2; 2 mM MgAc; 0.1 mM EDTA; 1 mM DTT; 0.5 mM PMSF) with 0.5% NP40 for 40 min on ice. The sample was fractionated after centrifugation at 600 × g for 15 min at 4℃. The supernatant was collected as cytoplasmic fraction. The precipitate was washed using sucrose buffer and centrifugated at 600 × g for 10 min at 4℃ three times. The precipitate was resuspended using RIPA buffer  supplemented with 1 mM PMSF and 1% phosphatase inhibitor (BOSTER), and used as nuclear fraction after sonication.Cells were collected and lysed with sucrose buffer  supplemented with 1 mM PMSF and 1% phosphatase inhibitor (BOSTER), and then sonicated with bath sonicator, centrifuged at 12,000 rpm for 10 min at 4 ℃. The supernatant of lysates was incubated with primary antibody and protein G agarose beads  at 4 ℃. The immunocomplexes were washed with ice-cold 1 × PBS four times and then boiled in the SDS sample buffer for 10 min. Both lysates and immunoprecipitates were identified by western blot.Cells were grown in glass base dishes. FRAP assay was conducted using the FRAP module of the NIKON N-SIM E confocal microscopy system. Proteins with fluorescent label GFP and RFP were bleached using a 488 nm and a 555 nm laser beam separately. Bleaching was focused on a circular region of interest (ROI) using 70% laser power (3 s) and time-lapse images were collected. Fluorescence intensity was measured using Image J software.Cells were cultured on coverslips, fixed in 4% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X‐100 (PBS) for 5 min, blocked with 1% (w/v) BSA (PBS) for 1 h, and then incubated with primary antibodies overnight. After washing, cells were incubated with fluorescence-labeled secondary antibody for 1 h at room temperature. The coverslips with cells were sealed with Fluor shield mounting medium with DAPI and imaged using a Super-Resolution Microscope . The number and size of p62 bodies were analyzed with the Image J software.The primary antibodies were used: Smurf1 , Keap1 , Keap1 , p62 , p62 , p62 , LC3B , LC3B , NBR1 , HA , Nrf2 , Flag .® 555 goat anti-mouse IgG . Alexa Fluor® 488 goat anti-rabbit IgG , Alexa Fluor® 555 goat anti-rabbit IgG , Alexa Fluor® 488 goat anti-mouse IgG , Alexa Fluor® 647 goat anti-mouse IgG , Alexa Fluor® 647 donkey anti-goat IgG , Alexa Fluor® 405 goat anti-rabbit IgG .The secondary antibodies were used: Alexa Fluor3) using a sterile razor blade and scissors. The chopped materials were incubated with 5 ml of trypsin EDTA for 30 min at 37 ℃ and then added 1 mL DMEM medium with 10% FBS to the chopped embryos, followed by centrifuging at 1000 rpm speed for 5 min. The pellets were resuspended and transferred to a 60 mm dish containing DMEM (with FBS and Penicillin/Streptomycin).Primary MEFs were obtained from embryos at 14-16 days post-coitum. The uterine horns of anesthetized pregnant mice were rinsed. The embryos were separated from placenta and placed in a 5 cm plate containing ice-cold sterile PBS. Remove the head, liver, and gut from the embryo and place the remaining portion in a 5 cm plate containing trypsin EDTA and cut into small pieces . Mice were housed in specific pathogen‐free facilities, and the Ethics Review Committee for Animal Experimentation of Beijing Institute of Technology University approved the experimental protocol.C699A, GFP-p62K7A/D69A and GFP-p62S349A site mutant were generated by the site-directed mutagenesis. p62K7A/D69A was amplified from GFP-p62K7A/D69A and then inserted into RFP vector. Full-length Nrf2 was amplified from pCDNA3-Myc3-Nrf2  and then inserted into RFP vector. Full-length NBR1 was amplified from pMXs-IP GFP-NBR1  and then inserted into PKH3-3 × HA and pEGFP-N3 vector.Full-length Smurf1 cDNA was amplified and then separately inserted into PKH3-3 × HA, 3 × Flag, pEGFP-N3, RFP and PGEX-5X-1 vector. Similarly, full-length p62 cDNA was amplified and then inserted into RFP-GFP, pEGFP-N3 and RFP vector. Full-length LC3 was amplified from RFP-GFP-LC3  and then inserted into PET-21a-Flag vector. Smurf1-C2, Smurf1-ΔC2, Smurf1-WW, Smurf1-HECT, Smurf1-ΔHECT was amplified from Flag-Smurf1 and then separately inserted into 3 × Flag. Flag-Smurf1-CS, Flag-Smurf1-ΔWW, Flag-Smurf1-CS-ΔWW, Flag-Smurf1His-Flag, His-Flag-LC3, or GST-Smurf1 protein is extracted from E. coli strain BL21 using ProBond™ Purification System  according to the manufacturer’s instructions. 20 μl of Flag beads  were incubated with His-Flag or His-Flag-LC3 overnight at 4 °C and then washed with ice-cold sterile PBS three times. The beads were incubated with GST-Smurf1 for 6 h at 4 °C followed by washing with ice-cold PBS four times, then boiled in 2 × loading buffer  for 10 min and identified by western blot.Additional file 1: Figure S1. Smurf1 promotes the formation of p62-liquid droplets. A LN229 cells were transfected with Flag or Flag-Smurf1, fixed followed by immunofluorescence stained with anti-Smurf1 and anti-p62 antibodies. The nuclear was stained with DAPI. Bar: 5 µm. B LN229 cells with Flag, Flag-Smurf1, or Flag-Smurf1C699A were fixed after treating with MG132 , then immunofluorescence stained with anti-p62 antibody. Bar: 5 µm. Bar graphs indicate the number and size of p62 puncta in each cell . The nuclear was stained by DAPI. NS p > 0.05, * p < 0.05, *** p < 0.001 as determined by unpaired two-tailed Student’s t-test. C LN229 cells cultured in glass-bottom plates were transfected with GFP-p62. Bar: 1 µm. The signal recovery after photobleaching was measured; mean ± SD, n = 20 droplets examined over three independent experiments. D LN229 cells cultured in glass-bottom plates were transfected with GFP-Smurf1 and treated with MG132 . Bar: 1 µm. The signal recovery after photobleaching was measured; mean ± SD, n = 20 droplets examined over three independent experiments. Figure S2. p62 phase separation is required for Smurf1-mediated Nrf2 activation. A LN229 cells were transfected with control or Smurf1 siRNA oligos. Cell lysates were prepared and subjected to western blot analysis with the indicated antibodies . The right panels show relative intensity of p62 and Nrf2 in total cell (mean ± SD from 3 independent experiments). ** p < 0.01 as determined by unpaired two-tailed Student’s t-test. B 293T cells were transfected with control or Smurf1 siRNA oligos. Cytosolic and nuclear fractions were prepared and subjected to western blot analysis with the indicated antibodies . C LN229 cells were transfected with control or Smurf1 siRNA oligos. Total RNAs were prepared from these LN229 cells. The relative mRNA levels of Smurf1 and NQO1 were performed by qRT-PCR analysis. Values were normalized against the amount of mRNA in LN229 transfected with control siRNA oligos; mean ± SD from 3 independent experiments. *** p < 0.001 as determined by unpaired two-tailed Student’s t-test. D LN229 cells treated with control or p62 siRNA oligos were transfected with GFP, GFP-p62, or GFP-p62K7A/D69A. Cell lysates were prepared and subjected to western blot analysis with the indicated antibodies . Figure S3. Smurf1 increases the binding affinity between p62 and Keap1 to activate Nrf2. A LN229 cells were transfected with control or Smurf1 siRNA oligos. Cell lysates were prepared and subjected to western blot analysis with the indicated antibodies . Bar graphs indicate the quantitative densitometric analysis of the indicated proteins relative to β-actin. Values were normalized against the intensity of LN229 transfected with si-Control; mean ± SD from 3 independent experiments. * p < 0.05, *** p < 0.001 as determined by unpaired two-tailed Student’s t-test. B LN229 cells treated with control or PTEN siRNA oligos were treated by treated by control or Smurf1 siRNA oligos. Cell lysates were prepared and subjected to western blot analysis with the indicated antibodies . C LN229 overexpressed with Flag or Flag-Smurf1 were transfected with HA or HA-PTEN, fixed after treating with MG132  and then performed by immunofluorescence analysis with anti-p62 antibody. The nuclear was stained with DAPI. Bar: 5 µm. Bar graphs indicate the number and size of p62 puncta in each cell . NS p > 0.05, *** p < 0.001 as determined by unpaired two-tailed Student’s t-test. D LN229 cells were transfected with Flag or Flag-Smurf1, fixed after treating with MG132 , and then immunofluorescence stained with anti-Keap1 and anti-p62 antibodies. The nuclear was stained by DAPI. Bar: 5 µm. Bar graphs indicate the number of Keap+p62+ puncta in each cell . *** p < 0.001 as determined by unpaired two-tailed Student’s t-test. E LN229 cells overexpressed GFP-p62 were fixed after transfecting with Flag or Flag-Smurf1, and then immunofluorescence stained with anti-Keap1 and anti-Flag antibodies. The nuclear was stained with DAPI. Bar: 5 µm. F LN229 cells were transfected with control, Smurf1 or p62 siRNA oligos, fixed after treating with MG132 , and then immunofluorescence stained with anti-p62 and anti-Keap1 antibodies. The nuclear was stained by DAPI. Bar: 5 µm. Bar graphs indicate the number of Keap+p62+ puncta in each cell . NS p > 0.05, * p < 0.05 as determined by unpaired two-tailed Student’s t-test. G LN229 cells were transfected with control or p62 siRNA oligos, fixed after treating with MG132 , and then immunofluorescence stained with anti-Keap1, anti-p62, and anti-Smurf1 antibodies. The nuclear was stained by DAPI. Bar: 5 µm. Figure S4. NBR1 enhances the Smurf1 mediated p62 liquid-droplets accumulation. A LN229 cells were transfected with control or Smurf1 siRNA oligos. Cell lysates were prepared and subjected to western blot analysis with the indicated antibodies . Bar graphs indicate the quantitative densitometric analysis of the indicated proteins relative to β-actin. Values were normalized against the intensity of LN229 transfected with si-Control; mean ± SD from 3 independent experiments. NS p > 0.05, * p < 0.05, ** p < 0.01 as determined by unpaired two-tailed Student’s t-test. B LN229 cells treated with control or Smurf1 siRNA oligos were transfected with Flag or Flag-Smurf1, fixed after treating with MG132 , and then immunofluorescence stained with anti-NBR1 and anti-p62 antibodies. The nuclear was stained by DAPI. Bar: 5 µm. Bar graphs indicate the number and size of NBR1+p62+ puncta in each cell . * p < 0.05, ** p < 0.01 as determined by unpaired two-tailed Student’s t-test. C LN229 cells treated with GFP-p62 cultured in glass-bottom plates were transfected with control, Smurf1, or NBR1 siRNA oligos. The signal recovery after photobleaching was measured; mean ± SD, n = 20 droplets examined over three independent experiments. Figure S5. Smurf1 mediated NBR1 expression in p62 dependent manner A LN229 cells were transfected with control, Smurf1, p62 or NBR1 siRNA oligos. Cell lysates were prepared and subjected to western blot analysis with the indicated antibodies . B LN229 cells treated with control or Smurf1 siRNA oligos were transfected with Myc or Myc-Nrf2. Cell lysates were prepared and subjected to western blot analysis with the indicated antibodies . C LN229 cells were treated with control or Smurf1 siRNA oligos and overexpressed Myc or Myc-Nrf2, fixed after treating with MG132 , and then immunofluorescence stained with anti-NBR1 and anti-p62 antibodies. The nuclear was stained by DAPI. Bar: 5 µm. Bar graphs indicate the number of NBR1+p62+ puncta in each cell . * p < 0.05, *** p < 0.001 as determined by unpaired two-tailed Student’st-test. D LN229 cells treated with control, NBR1, Nrf2, or p62 siRNA oligos were transfected with HA or HA-NBR1, and then overexpressed Flag or Flag-Smurf1. Total RNAs were prepared from these LN229 cells. Bar graphs indicate the amount of NQO1 mRNA. Values were normalized against the amount of NQO1 mRNA in LN229 transfected with control siRNA oligos, HA, and Flag; mean ± SD from 3 independent experiments. NS p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001 as determined by unpaired two-tailed Student’s t-test. E LN229 cells treated with NBR1 or p62 siRNA oligos were transfected with GFP, GFP-p62WT, GFP-p62S349A or GFP-p62K7A/D69A, and overexpressed Flag, Flag-Smurf1, or HA-NBR1. Total RNAs were prepared from these LN229 cells. Bar graphs indicate the amount of NQO1 mRNA. Values were normalized against the amount of NQO1 mRNA in LN229 transfected with p62 siRNA oligos, HA-NBR1, GFP, and Flag; mean ± SD from 3 independent experiments. NS p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001 as determined by unpaired two-tailed Student’s t-test. Figure S6. NBR1 enhances Smurf1-drived Nrf2-mediated oxidative stress response. A LN229 cells transfected with control or p62 siRNA oligos were overexpressed Flag or Flag-Smurf1, and treated with control (PBS) or H2O2  after transfecting with control or NBR1 siRNA oligos. Cell lysates were prepared and subjected to western blot analysis with the indicated antibodies . B LN229 cells transfected with control or p62 siRNA oligos were overexpressed Flag or Flag-Smurf1, and treated with control (PBS) or H2O2  after transfecting with control or NBR1 siRNA oligos. Total RNAs were prepared from these LN229 cells. Bar graphs indicate the amount of NQO1 mRNA. Values were normalized against the amount of mRNA in LN229 transfected with control siRNA oligos and Flag; mean ± SD from 3 independent experiments. NS p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001 as determined by unpaired two-tailed Student’s t-test.


Moreover, the phosphorylation of the transcription factors CREB and ATF1, that are MSK physiological substrates, and the expression of the CREB-dependent gene encoding DUSP1, were impaired in p38δ-deficient macrophages. Also, the transcription of IL-1Ra mRNA, that is MSK-dependent, was reduced. Our results indicate that MSK activation can be one possible mechanism by which p38δ regulates the production of a variety of inflammatory molecules involved in immune innate response.Mitogen- and Stress-activated Kinase (MSK) 1 is a nuclear protein, activated by p38α Mitogen-Activated Kinase (MAPK) and extracellular signal-regulated kinase (ERK1/2), that modulate the production of certain cytokines in macrophages. Using knockout cells and specific kinase inhibitors, we show that, besides p38α and ERK1/2, another p38MAPK, p38δ, mediates MSK phosphorylation and activation, in LPS-stimulated macrophages. Additionally, recombinant MSK1 was phosphorylated and activated by recombinant p38δ, to the same extent than by p38α, in  Toll-like receptor (TLR) signalling is fundamental in the recognition of pathogen-associated pattern molecules in innate immune cells. TLR activation will trigger the secretion of inflammatory cytokines and other pro-inflammatory mediators, which function is to eliminate infectious pathogens . The prop38MAPKs encompasses an important group of kinases that belong to the MAPK family, together with Extracellular signal-Regulated Kinase 1/2 (ERK1/2), c-Jun N-terminal Kinases (JNKs) and ERK5. There are four p38MAPKs, p38α, p38β, p38γ and p38δ, encoded by different genes . p38γ anin vitro. We found that in macrophages p38δ is involved in MSK1 phosphorylation and activation, and as a result, in the phosphorylation of the transcription factor CREB and in the transcriptional induction of CREB-dependent immediate early genes such as the dual-specificity phosphatase 1 (DUSP1) or the IL-1 receptor antagonist (IL-1Ra). All these data suggest that p38δ regulates the production of different anti-inflammatory molecules by controlling the activation of the MSK-CREB axis and plays an important role in macrophages during the innate immune and inflammatory response.Here we analysed how the lack of p38γ and/or p38δ affects the activation of different signalling pathways in macrophages stimulated with the TLR4 ligand, the endotoxin lipopolysaccharide (LPS), and report that the activation of Mitogen- and Stress-activated Kinase-1 (MSK1) is impaired in p38δ- and p38γ/p38δ-null cells. MSK1, and the related MSK2, are nuclear kinases activated downstream of p38α and ERK1/2 that phosphorylate the transcription factor CREB . MSK1 anAntibodies against total ERK1/2 (#9102), phospho-ERK1/2 , total IκBα (#9242), total JNK1/2 (#9252), phospho-p38MAPK , total MK2 (#3042), phospho-MK2 , total MSK1 (#3489), phospho-MSK1 , phospho-c-Jun  and phospho-IKKα/β  were purchased from Cell Signaling Technology. Antibodies to Phospho-CREB  was from Millipore, anti-p38α (#sc-535) and anti-DUSP1 (#sc-373841) were from Santa Cruz, anti-active phospho-JNK1/2  from R&D System, and anti-α-Tubulin (#T9026) from Sigma. Secondary antibodies from Invitrogen  included Alexa Fluor 680 donkey α-sheep IgG (H + L) (#A21102), Alexa Fluor 680 goat α-rabbit IgG (H + L) (#A21109), Alexa Fluor 700 goat α-mouse IgG (H + L) (#A21036).Kinase inhibitors SB203580 (inhibits p38α/p38β ) was purhttps://mrcppureagents.dundee.ac.uk). pGEX4T-p38γD171A (GST-p38γKD), pGEX4T-p38δD168A (GST-p38δKD), pGEX4T-p38δ (GST-p38δ), pGEX6P-MSK1(GST-MSK1) and pGEX6P-CREB (GST-CREB) were from the DSTT, expressed in E. coli strain BL21 and purified as described in ; University of Dundee  . Adult mice 12-week-old C57BL/6J-WT, p38γ/δ−/−, -p38γ−/− and -p38δ−/− were used in this work.Bone marrow derived macrophages (BMDM) lacking p38γ, p38δ or p38γ/δ were obtained from adult mouse femur and tibia as described elsewhere . BrieflyFor mRNA expression analysis, BMDM were lysed with NZYol (NZYtech) and the RNA extracted using a standard protocol with chloroform-isopropanol-ethanol.p38MAPKs were assayed using Myelin Basic Protein (MBP) as substrate . BrieflyMSK1  was first activated with p38α or p38δ (0.5 U/mL) in kinase assay buffer  and 0.1 mM ATP. After the indicated times at 30°C, fractions containing active MSK1 were diluted 1:100 in kinase assay buffer plus 0.1 mM [γ32P]ATP (specific activity: ∼3 × 106 cpms) or 0.1 mM ATP, and GST-CREB . The reactions were carried out at 30°C for 15 min and terminated by adding 4 x SDS-PAGE sample buffer containing 1% (v/v) 2-mercaptoethanol. Reaction samples were electrophoresed and autoradiographed. Phosphorylated MSK1 and CREB were quantified using the Fiji program.Protein samples were resolved in sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes, blocked (30 min at 25°C) in 50 mM Tris/HCl pH 7.5, 0.15 M NaCl and 0.05% (v/v) Tween (TBST buffer) with 10% (w/v) dry milk. Then membranes were incubated in TBST buffer with 5% (w/v) dry milk and 0.5–1 μg/mL antibody (2 h at 25°C or overnight at 4°C). Proteins were detected using fluorescently labelled secondary antibodies and the Odyssey infrared Imaging System .cDNA for real-time quantitative PCR (qPCR) was generated from total RNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Real-time qPCR reactions were performed in triplicate as described  in MicroIL-1Ra:forward 5′-GGCAGTGGAAGACCTTGTGT andrevers 5′-CATCTTGCAGGGTCTTTTCC;β-actin:forward 5′-AAGGAGATTACTTGCTCTGGCTCCT andrevers 5′-ACTCATCGTACTCCTGCTTGCTGAT;DUSP1:forward 5′-TGGGAGCTGGTCCTTATTTATT andrevers 5′-GACTGCTTAGGAACTCAGTGGAA.p values < 0.05 were considered significant. Data are shown as mean ± SEM.Data were processed using Student’s t-test. In all cases, Signalling pathways activated in response to the Toll-like receptor 4 (TLR4) ligand lipopolysaccharide (LPS)  were anaMSK1 phosphorylation was reduced in p38δ−/− macrophages. Since MSK1 is an ERK1/2 and p38α substrate, but the activation of these two kinases was not affected in p38δ−/− macrophages in response to LPS, we hypothesised that p38δ could directly phosphorylate MSK1. Thus, we next examined if recombinant MSK1 was phosphorylated by active recombinant p38δ in in vitro kinase assay using Mg [γ32P]-ATP and p38α, p38β and p38γ as comparative controls. All p38MAPKs were used at the same specific activity towards myelin basic protein (MBP), which is a pan-p38MAPK substrate. GST-MSK1 was phosphorylated by p38α, p38β and p38δ, but not by p38γ . The ratin vitro, p38δ inactive mutant ), or p38γ inactive mutant  as control , through CREB phosphorylation . We founMSKs also regulate the transcription of the anti-inflammatory molecule, the IL-1 receptor antagonist (IL-1Ra) in macrophages . Consistin vitro and in macrophages and, on the basis of this, propose a new way of MSK activation in cells. It is well stablished that MSKs are regulated by multiple phosphorylation. Both p38α and ERK1/2 mediate MSK phosphorylation at T581 and S360 in cells. Of these, T581 is required for the activation of MSK C-terminal kinase domain, in response to cellular stress or mitogens, and causes the autophosphorylation in other MSK domains ; however, this is phosphorylated by p38δ in p38γ−/− MEFs, and by p38α in p38γ/δ−/− cells . Thus, tDUSP1 is a dual specificity phosphatase that inactivates p38α and JNK1/2 . In LPS-In addition to DUSP1 and IL-1Ra, MSKs regulate the expression of the anti-inflammatory cytokine IL-10 in macrophages. It has been shown that MSK-mediated phosphorylation of CREB at Ser133 is required for its binding to the promoter of IL-10 after LPS stimulation in BMDM. IL-10 production is inhibited in MSK1/2−/− BMDM compared to WT . AccordiIn summary, we show here that, even in the presence of fully active p38α and ERK1/2, p38δ is essential for MSK1 phosphorylation/activation in macrophages. Also, p38δ regulates MSK1 downstream targets and this could limit the inflammatory signalling pathway downstream of TLRs.


Renal ischemia-reperfusion (IR) injury can lead to acute kidney injury, increasing the risk of developing chronic kidney disease. We hypothesized that mild therapeutic hypothermia (mTH), 34 °C, applied during ischemia could protect the function and structure of kidneys against IR injuries in mice. In vivo bilateral renal IR led to an increase in plasma urea and acute tubular necrosis at 24 h prevented by mTH. One month after unilateral IR, kidney atrophy and fibrosis were reduced by mTH. Evaluation of mitochondrial function showed that mTH protected against IR-mediated mitochondrial dysfunction at 24 h, by preserving CRC and OX-PHOS. mTH completely abrogated the IR increase of plasmatic IL-6 and IL-10 at 24 h. Acute tissue inflammation was decreased by mTH (IL-6 and IL1-β) in as little as 2 h. Concomitantly, mTH increased TNF-α expression at 24 h. One month after IR, mTH increased TNF-α mRNA expression, and it decreased TGF-β mRNA expression. We showed that mTH alleviates renal dysfunction and damage through a preservation of mitochondrial function and a modulated systemic and local inflammatory response at the acute phase (2–24 h). The protective effect of mTH is maintained in the long term (1 month), as it diminished renal atrophy and fibrosis, and mitigated chronic renal inflammation. Renal ischemia-reperfusion (IR) injury can occur in various pathological situations such as resuscitated cardiac arrest, hemodynamic shock , surgery involving supra-renal aortic clamping , renal clamping , or renal transplantation . Renal IMild therapeutic hypothermia (mTH) is currently used in clinical practice for neuroprotection against IR injury , supportIt has been well-established that the mitochondrial permeability transition pore (mPTP) is a major determinant of IR injuries. The mPTP is therefore an interesting therapeutic target to protect from IR injuries . DelayinRenal IR is commonly associated with tissue and systemic inflammation . MoreoveHere, the objective of our work was to test the efficiency of mild hypothermia conducted only during the short period of renal ischemia. We hypothesized that this stimulus would inhibit mPTP opening in parallel with a modulation of the inflammatory response that would protect kidney function and structure against IR in the acute phase of reperfusion and during the remodeling in the chronic phase.Therefore, we conducted this study in mice with two different protocols: a bilateral renal IR model to assess acute injuries in the first 24 h, and a unilateral renal IR model, less severe to assess chronic lesions at 1 month. We aimed to confirm in those models that mTH (1) prevents AKI and CKD, (2) improves renal mitochondrial function, and (3) modulates the local and systemic inflammatory response in the short and long term.p = 0.58). No mortality was observed in the Sham-37 °C and Sham-34 °C groups during the 24 h of reperfusion. There was also no mortality in all the groups of mice that underwent 2 h of reperfusion.Protocol 1, 20 min of bilateral renal ischemia, was a model of severe acute IR kidney injury, and it aimed at addressing the effect of mTH in the first 24 h of reperfusion. In Protocol 1, 3 mice out of 9 (33%) died in the IR-37 °C group, versus 1 mouse out of 9 (11%) in the IR-34 °C group during the 24 h of reperfusion , and was even higher after 24 h of reperfusion (60.8 mmol/L [58.0–69.7] vs. 6.2 mmol/L [5.7–8.1] in IR-37 °C vs. Sham-37 °C respectively) (p = 0.004). When mTH was applied during ischemia, plasmatic urea concentration did not increase after 2 h of reperfusion (16.4 mmol/L [13.2–17.9]) when compared to Sham-34 °C (p = 0.06). Twenty-four hours after reperfusion, the plasmatic urea level concentration had significantly increased but remained lower in IR-34 °C mice than in IR-37 °C ones (p = 0.001)  allowed us to assess acute effects and injury of IR on kidneys. Kidney function was evaluated by plasma urea assay. Plasma urea increased in the IR-37 °C group (18.7 mmol/L [17.3–19.0]) as soon as 2 h after reperfusion compared to Sham-37 °C (11.7 mmol/L [10.7–14.2]) (= 0.001) a,b.p = 0.004 and p = 0.02 respectively). However, the histological score of ATN was significantly lower (−34%) in IR-34 °C when compared to IR-37 °C (p = 0.03) , as compared to their respective Sham-37 °C or -34 °C group (score 0.80 [0.65–0.85] and 1.00 [0.80–1.00] respectively) ( = 0.03) c,e.p = 0.06), and apoptosis was significantly lowered when hypothermia was applied in IR-34 °C  compared to IR-37 °C (p = 0.03)  compared to Sham-37 °C  ( = 0.03) d.p < 0.001 and p = 0.03 respectively). mTH in the IR-34 °C group was associated with a significant decrease of renal atrophy compared to IR-37 °C (p = 0.001). In our protocol, IR did not modify the contralateral control kidney lengths in IR-37 °C and IR-34 °C (where a possible compensative hypertrophy could have been expected) compared to the respective Sham, although the contralateral kidney in IR-37 °C were significantly bigger compared to those in IR-34 °C  when compared to their respective Sham-37 °C or Sham-34 °C  (IR-34 °C a.p = 0.001). When mTH was applied, renal fibrosis was significantly less important in IR-34 °C  compared to IR-37 °C (p < 0.001), and it was not different from Sham-34 °C  (p = 0.18) , significantly more extensive compared to the Sham-37 °C group  ( = 0.18) b,e.p = 0.02 and p = 0.02 respectively)  and the IR-34 °C  groups compared to Sham  (ctively) d.p = 0.001, and 0.65 [0.60–0.69] p = 0.01 respectively). One month after reperfusion, these differences disappeared, and RVR in IR-37 °C and IR-34 °C were back to their baseline values   was modified early or chronically, in Protocol 2. Doppler US was used to follow non-invasively modifications in RVR. We showed a significant rise in RVR in ischemic kidneys at 20 min of reperfusion, in both IR-37 °C and IR-34 °C groups (Resistive Index 0.81 [0.75–0.86] and 0.83 [0.69–0.88] respectively), compared to their own baseline values measured a week before the procedure (Resistive Index 0.61 [0.58–0.67]  = 0.10) c. There Our hypothesis was that the beneficial effect of mTH on kidney function and structure could involve an early preservation of mitochondrial functions after 2 and 24 h of reperfusion. Temperature alone had no effect on renal mitochondrial functions between the 2 Sham groups in terms of calcium retention capacity (CRC) and oxidative phosphorylation (OX-PHOS) .2+/mg prot [220–380] at 37 °C and 280 nmol Ca2+/mg prot [220–420]) at 34 °C compared to their respective Sham conditions (320 nmol Ca2+/mg prot [280–640] and 280 nmol Ca2+/mg prot [260–440]) (p = 0.24 and p = 0.67)) (2+/mg prot [0–130]) compared to the Sham-37 °C group (320 nmol Ca2+/mg prot [280–360]) (p = 0.004). Conversely, the CRC was preserved by mTH in the IR-34 °C group 24 h after reperfusion (260 nmol Ca2+/mg prot [210–320]), compared to Sham-34 °C (280 nmol Ca2+/mg prot [240–340]) (p = 0.67) and reached a significantly higher value than in IR-37 °C group (p < 0.001) ) a. Twenty< 0.001) b.p < 0.05) (p = 0.009 and p = 0.02 respectively) (p > 0.05). Complex I activity in state 4 (oxygen consumption after ADP has been entirely phosphorylated) was altered 2 h after reperfusion in the IR-37 °C group when compared to Sham-37 °C (p = 0.008) (p = 0.55). The same isolated effect of IR at 37 °C was observed on Complex I respiratory coupling index (RCI), an index of mitochondria coupling, at 2 h of reperfusion (Electron transport chain (ETC) complexes I, II, and IV OX-PHOS activities in state 3, that is maximal ADP-stimulated mitochondrial respiration, were monitored in kidney mitochondria after 2 h or 24 h of reperfusion. After 2 h of reperfusion, OX-PHOS of complexes I, II, and IV were lowered in the IR-37 °C group, compared to Sham-37 °C ( < 0.05) a,c,e. Atctively) b,d,f. Co= 0.008) . On the erfusion . No effeerfusion .Temperature alone in the two Sham groups had no influence on the plasmatic concentrations of cytokines, nor on their mRNA expression in kidneys . Becausep = 0.06), rather than IR-34 °C versus Sham-34 °C  (p = 0.03)  a. After  = 0.03) b.p = 0.004), whereas mTH in IR-34 °C was associated with an undetectable plasmatic IL-10, as in the Sham 34 °C group , which was lessened by half in the IR-34 °C group, making it non-significant when compared to Sham-34 °C (p = 0.06). Moreover, IL-6 mRNA increased expression was higher in the IR-37 °C group compared to the IR-34 °C group  (p = 0.004)  a. After = 0.004) b.p = 0.01) (There was no significant modification of TNF-α expression after 2 h of reperfusion c. Nevert = 0.01) d.p = 0.03). mTH avoided modification of IL-1β expression (p = 0.10)  e. After  = 0.10) f.Renal IR or mTH had no significant effect on mRNA expression of TGF-β, MCP1 or NFkB after 2 or 24 h of reperfusion .p < 0.05) (p = 0.004) (p = 0.009) (p = 0.01) even if it seemed to have the same profile at 37 °C and did not reach significance, possibly because of an outlier  a,c,e. Al= 0.004) b. On the= 0.009) c. MCP1 m outlier f. Neithe outlier d.p = 0.003). However, mTH prevented this macrophagic infiltration in IR-34 °C (0.74% [0.48–1.02] of examined area) compared to IR-37 °C (p = 0.005), and it was not different from Sham-34 °C  compared to Sham-37 °C (respectively 2.58% [1.21–5.19] and 0.34% [0.08–0.75] of examined area)  because it reflects more accurately clinical situations such as surgical procedures requiring a renal or aortic vascular clamping  or kidney harvesting for transplantation, where mTH would be the easiest to implement in a daily clinical practice. Per-conditioning with mTH seems to be currently its most effective use against IR injuries . In renaWe chose a core body temperature of 34 °C based on previous data obtained in the laboratory . mTH , but too severe to keep mice alive after 24 h. That is why we had to perform a milder procedure, Protocol 2, to allow for the survival of mice, while CKD effectively developed . Because these experimental protocols reproduced a good number of characteristics of the pathology studied, our models were pertinent, even if the use of two different models could nonetheless bring limitations. In Protocol 2, we only found a mild plasma urea elevation after renal IR, without differences between IR-37 °C and IR-34 °C . This waIn Protocol 1, mTH applied during ischemia was beneficial on kidney function, assessed by plasmatic urea concentration and kidney structural damage, assessed by histology and acute tubular necrosis study, as well as apoptosis quantification by TUNEL. This is in line with previous work done by Xia et al.  in mice 2 saturation measured with photoacoustic imaging ) . All anin = 5), mTH Sham (Sham-34 °C) group (n = 4), normothermic ischemia (IR-37 °C) group (n = 13), and mTH ischemia (IR-34 °C) group (n = 12) (In Protocol 2 (1 month of reperfusion), mice were assigned to 4 groups—normothermic Sham (Sham-37 °C) group ((n = 12) . The proTwo mice were excluded from Protocol 1: one in the IR-37 °C group because the renal ischemia was not obtained  and one in the IR-34 °C group because a homeothermic pallet unit failure maintained involuntarily the core body temperature above 37 °C resulting in an early post-operative death of the mouse secondary to hyperthermia and dehydration.The operators performing all the procedures were blinded to the animals’ group assignment.Acute kidney injury was assessed 2 and 24 h after reperfusion by measuring the plasmatic concentration of urea. Urea was measured by a kinetic UV urease method  according to manufacturer’s instructions.Renal tissue samples were fixed in formaldehyde 2 h, 24 h and 1 month after reperfusion, and embedded in paraffin.To evaluate acute tubular necrosis, 4-μm sections were stained with periodic acid-Schiff reagents. Tubular injury was scored by a blinded pathologist semi-quantitatively who examined at least 10 fields, by using a scoring system ranging from 0 to 4. The lesions of ischemia were defined as a tubular dilatation, naked tubular basement membrane according to the following scoring system as described previously : 0, no tIn order to assess chronic renal fibrosis 1 month after reperfusion, 4-μm sections were stained with Masson’s trichrome. Images were analyzed with Fidji software , with a self-made macro tool allowing the automatic quantification of fibrosis based on the ratio between green pixels (representing fibrosis) and all the pixels representing the whole kidney biopsy, expressed as a percentage.Renal apoptosis was also quantified with terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining at 24 h and 1 month of reperfusion. Four-μm sections were stained by using a commercial kit , according to manufacturer’s instructions. Images were obtained by using a 20× objective on a Zeiss Olympus BX63 microscope and analyzed with Fidji software . Results were expressed as a percentage of the total number of cells (stained with DAPI) per 20× magnification field.At 1 month of reperfusion, renal tissue samples were also included in OCT and immediately frozen at −80 °C in order to perform latter immunofluorescence analysis.The operators performing all the procedures were blinded to the animals’ group assignment.For mitochondrial preparation, a half-kidney was excised and immediately placed in cold isolation buffer . The procedure was the same as was previously described . We used2+ necessary for the massive Ca2+ release by the mitochondria into the cytosol. Extra-mitochondrial Ca2+ concentration was measured with a Hitachi F2500 or F7000 spectrofluorometer as described before /peak systolic velocity), as the reflect of renal vascular resistance and renal injury .Circulating IL-6 and IL-10 concentrations were assessed on plasma by using enzyme-linked immunosorbent assay  according to manufacturer’s instructions.The expression of IL-6, IL-1β, TNF-α, TGF-β, MCP1, and NFkB mRNA was assessed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Prior to RNA isolation, snap frozen mouse kidney tissues were homogenized thanks to Precellys 24 . Total RNA was isolated from theses tissue homogenates, by using TRIzol  according to the manufacturer’s protocol and reverse-transcribed by using a PrimeScript RT reagent kit . Real-time PCR was performed by using the CFX96 real-time PCR system  and TB Green detection  according to the manufacturer’s instructions. The relative quantitation of gene expression was calculated by using CFX Manager  thanks to the Pfaffl method . ExpressImmunofluorescence was performed on 8 µm-sections of frozen renal tissue samples. Immunostaining was performed by using rat anti-mouse F4/80 antibodies  at 1:100 dilution. The secondary antibody used at 1:300 was Alexa Fluor 594-conjugated chicken anti-rat IgG . Briefly, the sections were fixed for 20 min with 3.7% formaldehyde, post-fixed with 0.1% Triton PBS solution, blocked with 2% goat serum for 30 min, and then incubated overnight with the primary antibody at room temperature. The sections were washed 3 times for 10 min each with PBS, followed by incubation for 30 min with the secondary antibody at room temperature. The sections were washed again with PBS, and nuclei were stained with DAPI. Images were obtained by using a 20× objective on a Zeiss Olympus BX63 microscope and analyzed with Fidji software . Results were expressed as a percentage of kidney tissue area per 20× magnification field.p value less than 0.05 was considered significant.Data are expressed as median [with interquartile range]. All groups were compared with a Kruskal–Wallis test. Comparisons between 2 groups was performed using Mann–Whitney tests. Paired data were compared with Wilcoxon matched-pairs signed rank test when appropriate . We showed that mTH (34 °C) applied during a renal ischemia, alleviates renal dysfunction and histological damage, through a preservation of mitochondrial function and a modulated systemic and local inflammatory response at the acute phase (2 to 24 h after reperfusion) by decreasing systemic and local IL-6, by increasing TNF-α mRNA expression and by mitigating IL-β1 mRNA expression. The protective effect of mTH is maintained in the long-term (1 month after reperfusion), as it diminishes renal atrophy and fibrosis, and still modulates the local renal inflammation, with mitigated TGF-β and activated TNF-α mRNA expressions.


In the crystal, N—H⋯N hydrogen bonding results in chains parallel to [010].The title complex exhibits a distorted octahedral coordination geometry. The CuN 9H5N4O)2(C10H9N3)2], was synthesized solvothermally. The complex exhibits a distorted octahedral coordination geometry. The CuII atom is located on an inversion centre. The distorted octahedral CuN6 coordination sphere is composed of bidentate 2,2′-dipyridylamine in the equatorial sites while the axial sites are occupied by 1,1,3,3-tetracyano-2-ethoxypropenide ligands. In the crystal, N—H⋯N hydrogen bonding results in chains parallel to [010].The title compound, [Cu(C The mixture was sealed in a Teflon-lined autoclave and held at 438 K for 2 d, and then cooled to ambient temperature at a rate of 10 K per hour (yield 42%). Green blocks of the title complex suitable for single-crystal X-ray diffraction were selected directly from the synthesized product.The title compound was synthesized solvothermally under autogenous pressure using a mixture of copper(II) sulfate pentahydrate , 2,2′-dipyridylamine  and potassium 1,1,3,3-tetracyano-2-ethoxypropenide  in water-methanol  global, I. DOI: 10.1107/S2414314622011804/bt4129Isup2.hklStructure factors: contains datablock(s) I. DOI: 2225624CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The authors apologise that parts of the data presented in RSC Advances paper.The authors have repeated the experiments to provide replacement data for (1) The original version of (2) The corrected version of 3O4 occurs at 588 cm−1. Other typical peaks could be assigned as follows, ν: 3440 cm−1; ν: 2949 cm−1, 2843 cm−1; ν scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>O): 1726 cm−1; δ(–CONH–): 1639 cm−1; δ(N–H): 1570 cm−1.”Page 63496 (right column), paragraph 2: “As shown in −1 shifted to 1533 cm−1, indicating the formation of hydrogen bond between 2,4,6-TCP and –NH2 groups on GO-Fe3O4@P.23 It was worth noting that the peaks at 1452 cm−1 and 1392 cm−1, due to the skeletal vibration of aromatic CC bonds, shifted to 1431 cm−1 and 1362 cm−1, respectively, confirming the π–π stacking interactions were formed between the benzene ring of 2,4,6-TCP and the hexagonal skeleton of the GO sheet of GO-Fe3O4@P.24”Page 63498 (left column), paragraph 2: “Besides, the characteristic peak of N–H bond at 1570 cm(3) The corrected version of 3O4@P could be deconvoluted into two individual peaks at binding energies of 400.7 eV and 399.3 eV, which could be assigned to N–H, and C–N, respectively. After 2,4,6-TCP adsorption, the binding energy of N–H bond shifted to 400.4 eV, which may be due to the formation of hydrogen bond between 2,4,6-TCP and the amine groups on the surface of GO-Fe3O4@P.”Page 63498 (left column) paragraph 2: “The N 1s high-resolution scan of GO-Fe3O4@P were quenched a lot after 2,4,6-TCP adsorption, implying the π–π stacking interaction appeared between 2,4,6-TCP and GO-Fe3O4@P.”Page 63498 (left column) paragraph 2: “The results indicated that the fluorescence intensities at ∼309 nm of GO-FeThe Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers.


Radiodynamic therapy (RDT) holds the potential to overcome the shallow tissue penetration issue associated with conventional photodynamic therapy (PDT). To this end, complex and sometimes toxic scintillator–photosensitizer nanoconjugates are often used, posing barriers for large-scale manufacturing and regulatory approval.Herein, we report a streamlined RDT strategy based on CsI(Na)@MgO nanoparticles and 5-aminolevulinic acid (5-ALA). 5-ALA is a clinically approved photosensitizer, converted to protoporphyrin IX (PpIX) in cancer cells’ mitochondria. CsI(Na)@MgO nanoparticles produce strong ~ 410 nm X-ray luminescence, which matches the Soret band of PpIX. We hypothesize that the CsI(Na)@MgO-and-5-ALA combination can mediate RDT wherein mitochondria-targeted PDT synergizes with DNA-targeted irradiation for efficient cancer cell killing. Because scintillator nanoparticles and photosensitizer are administered separately, the approach forgoes issues such as self-quenching or uncontrolled release of photosensitizers.When tested in vitro with 4T1 cells, the CsI(Na)@MgO and 5-ALA combination elevated radiation-induced reactive oxygen species (ROS), enhancing damages to mitochondria, DNA, and lipids, eventually reducing cell proliferation and clonogenicity. When tested in vivo in 4T1 models, RDT with the CsI(Na)@MgO and 5-ALA combination significantly improved tumor suppression and animal survival relative to radiation therapy (RT) alone. After treatment, the scintillator nanoparticles, made of low-toxic alkali and halide elements, were efficiently excreted, causing no detectable harm to the hosts.Our studies show that separately administering CsI(Na)@MgO nanoparticles and 5-ALA represents a safe and streamlined RDT approach with potential in clinical translation.The online version contains supplementary material available at 10.1186/s12951-022-01537-z. The emiynthesis , resultiynthesis . This tuynthesis –39 or anynthesis  in the cThe current RDT strategy affords several benefits. First, there is no need to load PS onto CsI(Na)@MgO nanoparticles, forgoing issues such as self-quenching among PS molecules and uncontrolled release of them. CsI(Na)@MgO nanoparticles are synthesized through wet chemistry with excellent reproducibility and scalability. Second, CsI(Na) nanoparticles comprise no transition and lanthanide elements. After treatment, the nanoparticles are degraded to low-toxic alkali and halogen ions, which are safely excreted from the hosts. Third, the PDT component specifically targets mitochondria, which are 5–10 times more susceptible to PDT than plasma membrane and endosomes/lysosomes , 42 that2. After reaction, we collected the products by centrifugation and redispersed them in hexane. The yield is ~ 90%. As-synthesized CsI(Na) nanoparticles were cubic  is hygroscopic. To prevent fast degradation, we imparted a layer of MgO onto CsI(Na) nanoparticles via seed-mediated growth . We chosWe coated CsI(Na)@MgO nanoparticles with DSPE-PEG-COOH and DSPE-PEG-Folate (molar ratio 3:1) through thin-film hydration. The resulting CsI(Na)@MgO@DSPE-Folate nanoparticles, hereafter referred to as CIS@M-F, are readily dispersed in aqueous solutions. Dynamic light scattering (DLS) showed that the average hydrodynamic size of CIS@M-F was 190.2 ± 30.1 nm  nanoparticles . Uncoated CsI(Na) nanoparticles rapidly degraded, releasing > 90% of their iodine within 30 min Fig. b. CIS@M-1O2 production using singlet oxygen sensor green (SOSG) as a fluorogenic sensor. SOSG intensity was increased by more than 4-fold in solutions containing both CIS@M-F and PpIX , and irradiated the solutions by X-rays (5 Gy). We observed a significant drop of 664 nm absorbance Fig. c, suggesWe first studied CIS@M-F uptake by 4T1 cells, in which folate receptor is upregulated. To this end, we labeled CIS@M-F with rhodamine-B. For comparison, we also prepared rhodamine-B-labeled CsI(Na)@MgO nanoparticles coated with DSPE-PEG-COOH only (referred to as CIS@M-C). Flow cytometry found significantly increased median fluorescence intensity (MFI) of rhodamine-B in cells treated with CIS@M-F relative to those treated with CIS@M-C Fig. a. Extend−1) and 5-ALA (200 µg mL−1) for 3 h, and then treated with ionizing radiation . We chose this drug-radiation interval because the 5-ALA-to-PpIX conversion peaked at ~ 3 h . Pre-treatment with CIS@M-F or a combination of CIS@M-F and 5-ALA moderately increased ·OH levels. The increase is attributed to high-Z element effects, which was observed with other nanoparticles   (DSPE-PEG(2000) Carboxylic Acid) , 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[folate(polyethylene glycol)-2000]  DSPE-PEG(2000) Folate , methylene blue , phosphate buffer saline , Milli-Q Water .Cs2CO3 was mixed with 10 mL 1-octadecene and 1 mL oleic acid in a 100 mL flask. The solution was heated to 150 °C and maintained at this temperature for 30 min with magnetic stirring. Next, 1 mL oleylamine, 0.02954 g 1,2-hexadecanediol, and 0.01 g NaI were added into the mixture. After reacting for 10 min, the solution was cooled to room temperature, and 0.3165 g I2 were added. The flask was then sealed and stirred for another 3 h. CsI nanoparticles were collected by centrifugation and washed with a 1:1 hexane and ethanol mixture 3 times.0.203 g Cs2, 0.053 g 1,2-tetradecanediol, and 20 mL benzyl ether in a 100 mL three-neck flask. The solution was heated to 120 °C under Argon protection and maintained at this temperature for 20 min. The solution was further heated to 310 °C before the addition of 1 mL pre-heated oleic acid. The reaction continued for 10 min before being cooled to room temperature. The product was collected by centrifugation and washed with a 1:1 hexane and ethanol mixture 3 times.In a typical reaction, 20 mg CsI(Na) nanoparticles in 5 mL hexane was mixed with 0.02 g Mg(acac)−1) and 25 µL DSPE-PEG(2000)-Folate in chloroform (10 mg mL−1) were added. The mixture was stirred at room temperature overnight, and the solvent was removed by rotary evaporation. The nanoparticles were dispersed in PBS and passed through a desalting column before use.20 mg CsI(Na)@MgO nanoparticles were dispersed in 2 mL chloroform. Into the solution, 75 µL of DSPE-PEG(2000)-COOH in chloroform (10 mg mL−1) and 25 µL DSPE-PEG(2000)-Folate in chloroform (10 mg mL−1) together with 25 µL of 16:0 Liss Rhod PE in chloroform (10 mg mL−1) were added. The mixture was stirred at room temperature overnight, and the solvent was removed by rotary evaporation. The nanoparticles were dispersed in PBS and passed through a desalting column before use.20 mg CsI(Na)@MgO nanoparticles were dispersed in 2 mL chloroform. Into the solution, 50 µL of DSPE-PEG(2000)-COOH in chloroform  which was equipped with an EDX system and Transmission Electron Microscope (FEI Tecnai20 and FEI Tecnai G2 F30 Hi-Res TEM). Nanoparticle composition was analyzed by Inductively Coupled Plasma Atomic Emission Spectroscopy using an Xseries II ICP/MS system (Thermo Electron Corporation). An iodide-selective electrode was used to conduct release experiments in PBS solutions of nanoparticles at room temperature (Mettler Toledo perfectION™).Nanoparticle crystallinity was assessed using the Bruker D8-Advance X-ray diffraction (XRD) diffractometer with Cu Kα radiation (λ = 1.5418 Å) at a scanning rate of 10° min−1 CIS@M-F, 0.04 M PpIX, and 100 µg mL−1 CIS@M-F plus 0.04 M PpIX solutions were prepared, distributed into a 96-well plate, and irradiated with 5 Gy X-ray (X-RAD 320). 80 µL 1 µм methylene blue was added to each well immediately following radiation, and the plate was shaken and kept in darkness at room temperature for 5 min before testing. A UV–vis spectrometer was then used to record absorbances (664 nm).100 µL PBS control, 100 µg mL−1 of penicillin (ATCC). Cells were maintained in a humidified, 5% carbon dioxide (CO2) atmosphere at 37 °C.4T1 breast cancer cells were used for in vitro and in vivo studies. Cells were grown in RPMI1640 medium which was supplemented with 10% FBS and 100 units mL-1 for CIS@M-F;19.5, 78.1, 312.5, 1250 µg mL−1 for 5-ALA) were added to each well for another 24 h of incubation. The ATP kit solution was then added to each well, and a 96-well microplate reader was used to measure total luminescence. Cell viability was calculated as a percentage of the luminescence of the untreated control.The ATP viability assay was performed according to the manufacturer’s protocol . 4T1 celThe ATP viability assay was performed according to the manufacturer’s protocol . 4T1 cells were seeded at 5000 cells/well in a white 96-well plate and incubated for 24 h before the addition of 20 µg 5-ALA to each well. Cells were incubated for 1 h before the addition of 10 µg CIS@M-F. Cells were then incubated for 2 h before being irradiated (5 Gy). The plate was returned to the incubator and maintained in darkness for 24 h before the ATP luminescence test.PpIX extraction was performed according to a published protocol . Cells w™ Cat#A36003) [ROS (reactive oxygen species) were measured with the APF assay (Invitrogen#A36003) . 4T1 cel™ Cat#S36002) [−1 CIS@M-F in 100 µL medium were added to each well. After 1 h, 20 µL 5-ALA (1 mg mL−1) was added. 5 Gy radiation was delivered following another 2 h of incubation. Fluorescence signals (ex/em: 504/525 nm) were recorded on a microplate reader (Biotek).The SOSG assay was conducted following the vendor’s protocol (Invitrogen#S36002) . 4T1 cel6 cells/well into 6-well plates. Then, nanoparticles were incubated with cells at a final nanoparticle concentration of 100 µg mL−1. Both CIS@M-F and CIS@M-C were labeled with Rhodamine B following a published protocol [Cell uptake of CIS@M-C and CIS@M-F nanoparticles were analyzed on a CytoFLEX flow cytometer. 4T1 cells were seeded at 0.5 × 10protocol . Followi−1 CIS@M-F and 200 mL−1 5-ALA were added to each well. 5 Gy X-ray irradiation was delivered after 4 h. Immediately following irradiation cells were washed with PBS three times and collected with a rubber scraper. Cell pellets were subjected to differential centrifugation at 4 °C and 12,000 rpm for 20 min to separate the mitochondrial and cytosolic fractions. Both the supernatant and mitochondrion were collected, aliquoted, sonicated and transferred into a 96-well plate. Test kit solution was added to each well, and the 96-well plate was shaken for 10 min in the dark at room temperature before measurement. Absorbance (450 nm) was measured on a microplate reader (Biotek).SOD activity was assessed following the vendor’s protocol . 4T1 cells were seeded into 6-well plates at 1 million cells/well. After 24 h, 2 mL RPMI medium containing 100 µg mL™ Cat#C10445) was used to assess lipid peroxidation. 4T1 cells were seeded into 96-well plates at 5000 cells/well. Following 24 h of incubation, 200 µg mL−1 5-ALA or 100 µg mL−1 CIS@M-F were added to each well. Following 4 h of incubation, the plate was irradiated (5 Gy). The Image-iT Lipid Peroxidation dye was added to each well, and the plate was incubated at 37 °C and 5% CO2 for 30 min. Green (ex/em: 488/510 nm) fluorescence intensity was used to quantitate lipid peroxidation.The Image-iT Lipid Peroxidation Kit (Invitrogen−1) and 5-ALA (200 µg mL−1) for 2 h prior to receiving 5 Gy X-ray irradiation. Control treatments included CIS@M-F, 5-ALA, or PBS. Following 24 h of incubation, cells were stained with the FAM-FLICA® Caspase-3/7 kit  following the manufacturer’s protocol. The caspase-3 activity was evaluated by measuring fluorescence signals (ex/em: 488/530 nm) on a microplate reader .4T1 cells were incubated with CIS@M-F (100 µg mL−1) for 2 h followed by 5-ALA (200 µg mL−1) for 3 h before receiving 5 Gy irradiation. Control treatments included CIS@M-F, 5-ALA, or PBS with or without irradiation. The medium was aspirated after 24 h, and cells were incubated in TMRE staining solution for 15 min. Fluorescence signals (ex/em: 549/575 nm) were measured on a microplate reader.Mitochondrial potential was assessed using the TMRE staining kit following the vendor’s protocol (Abcam Cat#ab113852). 4T1 cells were incubated with CIS@M-F (100 µg mL−1) for 2 h followed by 5-ALA (200 µg mL−1) for 3 h before receiving 5 Gy irradiation. Control treatments included CIS@M-F, 5-ALA, or PBS with or without irradiation. After 1 h, cells were collected, fixed, permeabilized, and stained with anti-rH2AX antibodies following the vendor’s protocol. Fluorescent images were acquired on a Zeiss LSM 710 confocal microscope. ImageJ was used to count the number of foci per cell.DNA damage was evaluated using anti-rH2AX (Alexa 647 labeled) antibodies . Briefly, 4T1 cells were seeded onto a 4-well imaging chamber at a density of 10,000 cells/well and incubated for 24 h. After washing, the cells were incubated with CIS@M-F (100 µg mL−1) for 2 h followed by 5-ALA (200 µg mL−1) for 3 h before receiving 0, 1, 3, 5, 7, and 9 Gy of radiation. Treated cells were trypsinized, replanted in petri dishes (100 * 15 mm), and incubated at 37 °C with 5% CO2. After 14 days, cells were rinsed carefully with PBS, fixed in 2–3 mL of 6.0% glutaraldehyde solutions, and treated with 1 mL of 0.5% crystal violet. After 10 min, cells were rinsed with D.I. water and dried before colony counting. Colonies containing at least 50 stained cells were included in survival fraction (SF) calculations.Clonogenic assays were performed using a modified protocol . 4T1 cel5 cells in 50 µL PBS into the right flanks of 5–6-week old female BALB/c mice (Charles River). When tumor volume reached 50 mm3, the animals were randomly divided into 6 groups (n = 5) and received the following treatments (Day 1): PBS plus ionizing radiation (PBS + IR), 5-ALA with irradiation (5-ALA + IR), CIS@M-F plus irradiation (CIS@M-F + IR), PBS only (PBS), CIS@M-F plus 5-ALA, no irradiation (CIS@M-F + 5-ALA), or CIS@M-F plus 5-ALA plus irradiation (CIS@M-F + 5-ALA + IR). 5-ALA (50 mg kg−1 in PBS) was administered intraperitoneally. CIS@M-F in PBS (1.25 mg kg−1) were intratumorally injected 2 h after the 5-ALA injection. A 320 KV cabinet irradiator  was used to irradiate (3 Gy) tumors 1 h after CIS@M-F administration, while the rest of the animal body was protected with lead. Animals underwent two additional treatment sessions on Days 3 and 5. Tumor size and body weight were inspected every 3 days. Tumor dimensions were measured with a caliper. Tumor volume was estimated by calculating (length) × (width)2/2. Animals were euthanized after 22 days. Tumors were dissected and sliced for H&E and Ki67 staining. Organs including the heart, spleen, liver, brain, intestine, kidney, and lung were also harvested for H&E staining.Animal studies were performed according to a protocol (A2020 06-004-R1) approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Georgia. The animals were maintained under pathogen-free conditions. 4T1 tumors were established by subcutaneously injecting 2 × 10−1) or PBS (control) via the tail vein. All mice were sacrificed after 2 weeks. Blood was collected through cardiac puncture for complete blood count (CBC), BUN, and ALT measurements. Major organs, including the heart, spleen, liver, brain, intestine, kidney, and lung were harvested. Half of the tissues were weighted, homogenized, and digested in hot nitric acid. Supernatants were subjected to ICP-MS analysis to measure tissue concentrations of cesium and iodine in tissues (µg/g of tissue). The remaining tissues were fixed and sliced for H&E staining.5–6 week-old female BALB/c mice (Charles River) were intravenously injected with CIS@M-F  nanoparticles, SEM and EDX of of CsI(Na)@MgO nanoparticles, zeta potential of CIS@M-F and CIS@M-C, in vitro studies investigating 5-ALA conversion to PpIX, cytotoxicity of 5-ALA and CIS@M-F in the absence of IR, rH2AX staining results, clonogenic assay results, H&E staining of organ tissues as well as serum BUN and ALT levels after animals being i.v. administrated with CIS@M-F.


Scientific Reports 10.1038/s41598-018-26768-9, published online 26 June 2018Retraction of: multiple lanes within each of the Coomassie blue staining images in Figs. 3h, 6c, S1a and S2a;DNP blot images in Figs. 3h (H2O2 + pNaKtide group), S1a (both lanes in Y group and left lane in O + WD + P group) and S2a (OB and O groups);pSRC blot images in Figs. S2b (OB group) and 3i .The Editors have retracted this Article. After publication, concerns were raised regarding high similarity among the following:Figs. 3a (Control and H2O2 + pNaKtide) and S4a (Control and UV + pNaKtide);Fig. S3a ;Figs. S3a (H2O2 200 μM) and Fig. S5a (Ouabain 1 nM).Further investigation has found that there are unexplained similarities between images representing different groups in:The Editors therefore no longer have confidence in the presented data.Juan R. Sanabria and Nader G. Abraham agree to this retraction. Komal Sodhi, Jiang Liu, Rebecca Pratt, and Joseph I. Shapiro do not agree to this retraction. Alexandra Nichols, Amrita Mallick, Rebecca L. Klug, Xiaoliang Wang, Krithika Srikanthan, Perrine Goguet-Rubio, Athar Nawab, Megan N. Lilly, and Zijian Xie have not responded to any correspondence from the Editors about this retraction.


In vitro binding of 125I-labeled affibodies with and without scFv8D3 was evaluated by ELISA and autoradiography. Brain uptake and retention of the affibodies at 2 h and 24 h post injection was studied ex vivo in WT and transgenic (tg-Swe and tg-ArcSwe) mice. At 2 h post injection, [125I]I-Z5 and [125I]I-Z1 displayed brain concentrations of 0.37 ± 0.09% and 0.46 ± 0.08% ID/g brain, respectively. [125I]I-scFv8D3-Z5 and [125I]I-scFv8D3-Z1 showed increased brain concentrations of 0.53 ± 0.16% and 1.20 ± 0.35%ID/g brain. At 24 h post injection, brain retention of [125I]I-Z1 and [125I]I-Z5 was low, while [125I]I-scFv8D3-Z1 and [125I]I-scFv8D3-Z5 showed moderate brain retention, with a tendency towards higher retention of [125I]I-scFv8D3-Z5 in AD transgenic mice. Nuclear track emulsion autoradiography showed greater parenchymal distribution of [125I]I-scFv8D3-Z5 and [125I]I-scFv8D3-Z1 compared with the affibodies without scFv8D3, but could not confirm specific affibody accumulation around Aβ deposits. Affibody-scFv8D3 fusions displayed increased brain and parenchymal delivery compared with the non-fused affibodies. However, fast brain washout and a suboptimal balance between Aβ and mTfR1 affinity resulted in low intrabrain retention around Aβ deposits.Affibodies targeting amyloid-beta (Aβ) could potentially be used as therapeutic and diagnostic agents in Alzheimer’s disease (AD). Affibodies display suitable characteristics for imaging applications such as high stability and a short biological half-life. The aim of this study was to explore brain delivery and retention of Aβ protofibril-targeted affibodies in wild-type (WT) and AD transgenic mice and to evaluate their potential as imaging agents. Two affibodies, Z5 and Z1, were fused with the blood–brain barrier (BBB) shuttle single-chain variable fragment scFv8D3. The online version contains supplementary material available at 10.1007/s11095-022-03282-2. Several of our studies using radiolabeled antibodies have shown that less than 0.05% of the administered dose resides in the brain 2 h after njection –4. The Bnjection , 6–12. Tnjection , 14. 8D3njection .Alzheimer’s disease (AD) is the most common form of dementia, and the number of cases is projected to triple by the year 2050 . To dateStaphylococcus aureus  binding domain of protein A in s aureus . The affs aureus . The smat cancer –27.SYM73, is a high affinity  Aβ-monomer targeting affibody. Conjugated with an albumin-binding domain (ABD) to increase circulation time, ZSYM73-ABD, was able to both decrease the Aβ plaque load and reduce cognitive decline in an AD mouse model (SYM73-ABD and a ZSYM73-ABD fused with the antibody 8D3’s mTfR1-binding single chain variable fragment (scFv8D3). Increased concentrations in the CSF was shown for scFv8D3-ZSYM73-ABD after 24 h, whereas ZSYM73-ABD CSF-concentrations decreased over time, in correlation with its blood serum concentration profile  ligands based on antibodies, able to visualize intra-brain Aβ-pathology in vivo –36. A smin vivo , 37. Howin vivo . The aimAβ42cc_1-His6 and ZAβ42cc_5-His6 were generated toward protofibrils of Aβ42CC, an engineered mimic of wild-type Aβ42 protofibrils (described in ref. 39) (42CC protofibrils (KD = 1.7 ± 0.6 nM), determined by surface plasmon resonance, and also detected wild type Aβ42 protofibrils in ELISA  of 8D3 (see “Cloning and protein production”). The four affibody constructs are hereafter referred to as Z5, scFv8D3-Z5, Z1 and scFv8D3-Z1 in this study (Table NSSGTTAASGSSGGSSSGAC). The 8D3 scFv was linked via a 10 amino-acid linker (NGAPGGGGSTSC) to the N-terminus of either of the amyloid beta protofibril-binding affibody molecules Z1 and Z5, respectively. Genetic sequences for control affibody molecules Z1 and Z5 were cloned into the pET26b( +) vector (Novagen), introducing a C-terminal His6-tag. The resulting plasmids [Z1-His6] and pET26b( +)[Z5-His6] were sequence verified by Sanger sequencing .The genetic sequences for the scFv8D3-fusion proteins were cloned into the pQMCF1 vector  , containEscherichia coli BL21 Star (DE3) cells  for production, according to preciously described protocol  EBNALT 85 cells for production using the Icosagen QMCF technology . Cells wprotocol . Overnig125I stock solution . Thereafter, 5 μg Chloramine-T  in PBS was added to a final volume of 110 μl. The reaction was incubated for 90 s at room temperature, and quenched with 10 μg sodium metabisulfite (Sigma Aldrich). The product was diluted to 500 μl with PBS, and separated from free-iodine in a PBS-equilibrated NAP-5 size exclusion column . The product was eluted with a total volume of 1 ml PBS.All four affibodies were radiolabeled by direct iodination using the chloramine T method . Briefly125I stock solution (Perkin-Elmer) was added together with PBS to the iodination tube to a total volume of 40 μl. The tube was incubated for 6 min on a shaker at room temperature to activate the iodine. 10–20 μg affibody was prepared in Protein LoBind tubes (Eppendorf). Thereafter, 20 μl activated iodine was added to each affibody, followed by a 10 min incubation at room temperature on a shaker (600 rpm). After the incubation, the product was diluted with PBS to 500 μl and purified as described above.Pierce Pre-Coated Iodination Tubes  was used as an alternative to the Chloramine T method to achieve milder reaction conditions . First, 2SO4. The plates were analyzed at 450 nm in a spectrophotometer. Affibody and secondary antibody sample dilutions were made in ELISA incubation buffer .The affibody binding to Aβ protofibrils and mTfR1 before and after radiolabeling was assessed with indirect ELISA in comparison with di-scFv3D6-8D3 . In shorSandwich ELISA was used to determine concentrations of affibodies after radiolabeling. Half area 96-well plates (Corning Inc.) coated with 0.5 μg/ml Goat-Anti-Affibody IgG  were incubated overnight in at 4°C. After 1 h of blocking with 1% BSA in PBS, affibody samples were serially diluted from 250 nM, followed by incubation overnight at 4°C. The plates were incubated with HRP-conjugated anti-His-Tag antibody (Proteintech) for 1 h incubation on a shaker. The signal was developed and plates read as described above.in vivo retention of the affibody constructs: tg-Swe, harbouring the Swedish (APP KM670/671NL) APP mutation, and tg-ArcSwe with a combination of the Arctic (APP E693G) and the Swedish APP mutations (tg-ArcSwe), both maintained on a C57BL/6 background. The mutation in tg-Swe mice leads to increased production of Aβ, and a late onset (at 12 months) of Aβ pathology, followed by rapid progression. In tg-ArcSwe mice, increased Aβ production in combination with an aggregation-prone Aβ mutated species produces an earlier onset (at 6 months) of pathology with dense Aβ-plaques  following the legislation and regulations of the Swedish Animal Welfare Agency and European Communities Council Directive of 22 September 2010 (2010/63/EU).Two AβPP transgenic mouse models were used in this study to investigate the -plaques . These t125I]I-Z5 or [125I]I-Z1 (0.9 MBq/nmol), and 1 nM of [125I]I-scFv8D3-Z5 or [125I]I-scFv8D3-Z1 (3–4 MBq/nmol) in PBS for overnight incubation at 4°C. The sections were washed 3 × 15 min in cold 0.1% BSA-PBS-buffer, 20 s in dH2O, and dried under constant air flow at RT for 1 h, then exposed to a phosphor imaging plate  for 3 h. The plate was scanned and digitalized at 600 dpi in a Cyclone Plus phosphor imager (PerkinElmer). Images were converted with the “Royal” lookup table and the intensity was adjusted individually for each protein in ImageJ. Regions of interest (ROIs) were quantified using the Integrated Density (IntDen) measurement in triplicate brain sections.Cryosections, 20 μm, were prepared from brains of old (2 years) wild type and tg-ArcSwe mice and mounted on Superfrost Plus glass slides . The frozen sections were adjusted to room temperature for 1 h, and blocked with 1% BSA in PBS for 1 h. The sections were incubated in PBS for 5 min, followed by the addition 4 nM of [125I-labeled affibodies via the tail vein. The animals were injected with either 0.49 ± 0.20 MBq of [125I]I-Z5, 0.43 ± 0.18 MBq of [125I]I-scFv8D3-Z5, 0.76 MBq ± 0.13 MBq of [125I]I-Z1 or 0.88 ± 0.34 MBq of [125I]I-scFv8D3-Z1. To investigate the blood pharmacokinetic profiles, blood samples of 8 μl were collected from the tail vein at time points between 30 min after administration and euthanization, i.e. at 2 h or 24 h post injection. At euthanization, transcardial perfusion under isoflurane anesthesia with 40 ml saline for 2.5 min was used to remove blood from the brain and organs before isolation. Subsequently, the brain was harvested and dissected into the right hemisphere, left cerebrum and left cerebellum. The brain samples were immediately frozen at -80°C. Lung, liver, kidney, heart, pancreas, spleen, femoral bone, skull bone and submandibular gland (smg) were isolated to study the biodistribution of the radiolabeled affibodies. The radioactivity of all samples was measured with a γ-counter . Antibody concentrations were expressed as percent of injected dose per gram tissue (%ID/g) or percent of injected dose corrected for body weight (bw) of the animal (%ID/g/bw). The reported brain concentrations were measured in the cerebrum of the left hemisphere.Mice were intravenously injected with 125I]I-affibody-injected animals were sectioned sagittally (20 μm) with a cryostat  and mounted on Superfrost Plus glass slides (Menzel Gmboltion). Duplicate sections from each animal together with a standard of 125I with known radioactivity were exposed to a phosphor imaging plate  for 7 days. The plates were scanned in a Cyclone Plus phosphor imager (PerkinElmer) at 600 dots per inch.The frozen right hemispheres from [125I]I-affibody-injected mice were fixed in ice-cold MeOH for 10 min and washed 2 × 5 min in PBS. Double CD31/Aβ40 staining was performed on selected WT, tg-Swe and tg-ArcSwe brain sections by the following procedure:Sagittal brain cryosections from I-di-scFv3D6-8D3 displayed a high signal and visibly accumulated near Aβ-plaques (green) already 12 h post injection , i.e. in a similar range as previously reported , despite fast blood elimination. This is more than tenfold higher than brain concentrations previously observed for iodinated IgG antibodies (0.03%ID/g brain) (2 fragment by adsorptive transcytosis (AMT), which is slower and less specific than RMT .BBB-shuttles such as scFv8D3 have proven important to potentiate the delivery of biologicals to the brain , 35, 49.in vivo interaction due the high TfR1 affinity.This confirms that TfR1 mediated transport is a robust strategy for enhancing brain delivery of affibody molecules . Di-scFv125I]I-Z5 and [125I]I-Z1. In combination with a higher brain-to-blood ratio of scFv8D3-fused affibodies, these results suggest that the fused affibodies did penetrate the BBB and bind to an intrabrain target. In contrast, [125I]I-Z5 and [125I]I-Z1 may have been associated with the endothelium of the brain vasculature at the early time point but without actually entering the brain.Nuclear track emulsion autoradiography showed that 2 h after administration, the scFv8D3-fused affibodies were highly distributed to endothelial cells in brain capillaries, but also to the parenchyma. The non-fused affibodies, however, seemed to cluster in larger vessels. Importantly, at 24 h after injection, although moderate, the brain retention of the scFv8D3-fused affibody molecules was considerably higher than for [42CC and Aβ42wt protofibrils  at 24 h after administration.Supplementary file1 (PDF 1798 kb)Below is the link to the electronic supplementary material.