During every cell cycle, both the genome and the associated chromatin must be accurately replicated. Chromatin Assembly Factor-1 (CAF-1) is a key regulator of chromatin replication, but how CAF-1 functions in relation to the DNA replication machinery is unknown. Here, we reveal that this crosstalk differs between the leading and lagging strand at replication forks. Using biochemical reconstitutions, we show that DNA and histones promote CAF-1 recruitment to its binding partner PCNA and reveal that two CAF-1 complexes are required for efficient nucleosome assembly under these conditions. Remarkably, in the context of the replisome, CAF-1 competes with the leading strand DNA polymerase epsilon (Polϵ) for PCNA binding. However, CAF-1 does not affect the activity of the lagging strand DNA polymerase Delta (Polδ). Yet, in cells, CAF-1 deposits newly synthesized histones equally on both daughter strands. Thus, on the leading strand, chromatin assembly by CAF-1 cannot occur simultaneously to DNA synthesis, while on the lagging strand these processes may be coupled. We propose that these differences may facilitate distinct parental histone recycling mechanisms and accommodate the inherent asymmetry of DNA replication. During every cell cycle, a new copy of the genome is made. At the same time, genomic chromatin organization must be replicated to ensure faithful transmission of the parental epigenetic state to both daughter cells after cell division. Therefore, genome and chromatin replication are tightly coupled and regulated by the concerted action of several dozens of proteins. Errors in both processes affect cell function; they can derail developmental programs or cause diseases, such as cancer .Proliferating Cell Nuclear Antigen (PCNA) for their function. PCNA is an essential homotrimeric clamp that encircles newly synthesized double-stranded DNA, tethering the DNA polymerases to DNA. It is abundant at replication forks where, in addition to the DNA polymerases, it binds many other factors involved in genome replication, chromatin assembly and the response to stress and damage  helicase complex, DNA polymerases and regulatory factors . Two disd damage .bona fide histone chaperones that are recruited to the replisome   and nd ASF1) . These pnd ASF1) . Replicand ASF1) ,22, a prnd ASF1) ,23–27.PCNA Interacting Peptides (PIPs) present on the large CAF-1 subunit (Chromatin Assembly Factor-1 (CAF-1) is a key regulator of chromatin assembly during DNA replication . CAF-1 d subunit . While tin vitro reconstitution of the eukaryotic replisome. We find that CAF-1 recruitment to PCNA requires DNA and is modulated by histones. Two CAF-1 complexes bind PCNA and are necessary for PCNA-dependent nucleosome assembly. CAF-1 interaction with PCNA inhibits the activity of the leading-strand DNA polymerase Polϵ, but not of the lagging-strand polymerase Polδ. Yet, in cells, we show that CAF-1 deposits histones equally on the leading and lagging strands during DNA replication. Thus, our work reveals an unexpected difference in the crosstalk between CAF-1, PCNA and the two replicative polymerases, suggesting different mechanisms for the coupling of nucleosome assembly to DNA synthesis on the two daughter strands.Here we developed biochemical systems to study the crosstalk between CAF-1 and key components of the DNA replication machinery, combining our previous CAF-1 histone chaperone assays ,55 with Xenopus laevis histones and FEN1. Several proteins used in our study were expressed and purified as previously described. This includes PCNA  and refolded as in . The expression was incubated for 16 h and cells were harvested and resuspended in 30 mM HEPES pH 7.5, 0,5 mM EDTA, 10% glycerol, 200 mM NaCl, 1 mM DTT, 0,5 mM p-methylphenyl-sulfonyl fluoride (PMSF) in presence of COMPLETE EDTA-free protease inhibitor (Roche). Cells were lysed with sonication. DNA was precipitated with 0.5% of poly(ethyleneimine) (PEI) and the lysate was clarified by centrifugation for 30 min at 35000xg. RFCΔN was precipitated with 0.28 g/ml of AmSO4. The precipitates were collected by centrifugation at 12 000×g for 45 min. Pellets were resuspended in 50 ml of 30 mM HEPES pH 7.5, 0,5 mM EDTA, 10% glycerol + 1 mM DTT, 0.5 mM PMSF in presence of COMPLETE EDTA-free protease inhibitor. The lysate was next dialyzed (12–14 MWCO) against 2 liters of 30 mM HEPES pH 7.5, 0,5 mM EDTA, 10% glycerol + 100 mM NaCl, 1 mM DTT for 2 h. RFCΔN was injected on HiTrap SP HP 5ml column (Cytiva) equilibrated in buffer SP-A . The column was washed with 25 ml of SP-A buffer and RFC∆N was eluted in a gradient of SP-B buffer  along 60 ml. Fractions containing RFCΔN were analyzed by SDS-PAGE and were pooled together. RFCΔN was then mixed with 3 ml of nickel beads equilibrated in His-A buffer . Beads were washed with 100 ml of His-A buffer and RFCΔN was eluted with His-B buffer . Fractions containing RFC were concentrated and further purified on HiLoad 16/600 Superdex 200 in 20 mM HEPES 7.5, 200 mM NaCl, 10% glycerol, 1 mM TCEP, 0.05% ampholytes. RFCΔN was concentrated and stored at –80°C.Rosetta2 cells containing pBL481-RFCΔN from Peter Burgers  were groA600 = 1.8. Cells were placed at 25°C and RPA expression was induced with 0.3 mM IPTG for 3 h. Cells were harvested and resuspended in lysis buffer  in presence of COMPLETE EDTA-free protease inhibitor (Roche). Cells were sonicated and the lysate was clarified by centrifugation at 50 000×g for 50 min. The supernatant was recovered and injected on HisTrap HP 5 ml column equilibrated in lysis buffer. The column was washed with 50 ml of lysis buffer, 100 ml of His-A buffer , and 25 ml of lysis buffer respectively. RPA was then eluted in a gradient of His-B buffer  along 50 ml. Fractions containing RPA were pooled and diluted in 50 mM HEPES pH 7.5, 5% glycerol, 0.01% IGEPAL CA-630, 1mM TCEP to bring the salt concentration to 150 mM NaCl. RPA was next injected on HiTrap Heparin HP 1ml equilibrated in QA buffer . The column was washed with 20 ml of QA buffer and RPA was eluted in a gradient of QB buffer  along 40 ml. Fractions containing RPA were pooled together and injected on HiLoad 16/600 Superdex 200 and eluted in 50 mM HEPES pH 7.5, 300 mM NaCl, 5% glycerol, 0.01% IGEPAL CA-630, 1 mM TCEP. RPA was concentrated and stored at –80°C.Rosetta2 cells transformed with pRSF-Duet, RPA, a gift from Xiaodong Zhang , were grX. laevis FEN1 (S-form) D181A mutant was codon-optimized, synthesized , and ligated into the BamHI–XhoI sites of the pGEX6P-1 vector. BL21 (DE3) RIL cells transformed with pGEX6P-1-xlFEN1.S_DA were grown in 2.4 l of LB + ampicillin at 30°C until A600 = 0.7. Cells were placed at 18°C and FEN1_DA expression was induced with 0.5 mM IPTG for overnight. Cells were harvested and resuspended in 40 ml lysis buffer  in presence of COMPLETE EDTA-free protease inhibitor (Roche). Cells were sonicated and the lysate was clarified by centrifugation at 26 500×g for 20 min. The supernatant was recovered and added to Glutathione Sepharose 4B beads (Cytiva) equilibrated in lysis buffer. After incubation for 90 min, the beads were washed with 100 ml of wash buffer 1 . Washed the beads with 50 mL wash buffer 2 . Beads were resuspended in 2 ml wash buffer 2 and RPA was cleaved from the beads overnight at 4°C using 100 U PreScission protease. Concentrated FEN1_DA was injected on Superose 6 Increase 10/300 GL (Cytiva) and eluted in Superose 6 buffer . Fractions were selected and FEN1_DA was concentrated and stored at -80°C.The cDNA encoding the full-length Histones H2A–H2B (containing H2B-T112C) and H3–H4 (containing H4-E63C) were labeled with maleimide AlexaFluor-647 (AF647) or AlexaFluor-488 (AF488) respectively ,58, as iPCNA-C4S-K164C and PCNAK164C were labeled with Alexa Fluor 546. PCNA was diluted in labelling buffer  to a final concentration of 1mg/ml. A 10-fold excess of TCEP was added to PCNA to ensure that all cysteines are effectively reduced. PCNA was then incubated with a 10-fold excess of AlexaFluor546. The reaction was incubated for 2 h at room temperature, then quenched with 20 mM DTT final concentration for 30 min. Labelled PCNA was then concentrated and injected on a Superdex 75 increase 10/300 column to remove free dyes. PCNA was eluted in 20 mM HEPES pH 7.5, 125 mM NaCl, 1 mM TCEP. Fractions containing labelled PCNA were pooled and concentrated, and the protein was stored at –80°C.Single-stranded DNA oligos of different lengths were purchased from IDT, either desalted (unlabeled oligos) or HPLC-purified (Alexa Fluor 647-conjugated oligos). For each length  a forward oligo and a reverse oligo in reverse complement sequence were ordered. The 18mer and 33mer forward oligos included a 5’ Alexa Fluor 647 label. Forward and corresponding reverse oligos were mixed in a 1:1 stoichiometric ratio at 20 μM each (18mer and 33mer) or 40 μM each (43mer and 53mer) with a final of 20 mM HEPES pH 7.5 and 25 mM NaCl. The mixed oligos were annealed by heating up to 95°C for 3 min, and then slowly cooled to room temperature over several hours. Annealed DNA was stored at –20°C.Kd values were calculated using a one site binding curve with hill slope in GraphPad Prism. The 18bp data was fitted to a one site binding curve with a Hill coefficient constrained to 1.Native DNA-protein complexes were allowed to form in NA buffer: 25 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.02% Tween-20, 1 mM TCEP. Increasing amounts of CAF-1 (0–5 μM) were incubated in buffer for 10 minutes before addition of DNA (50 nM). Single stranded DNA oligos for 18, 33, 43 or 53 bp were purchased from IDT  and ATP (3 mM). Next, CAF-1 (5μM) was added to these reactions and incubated for 15 min at room temperature. Samples were next spun down for 5 min at 17 000×g before injection on Superose 6 increase 3.2–300 columns connected to an AKTA pure system fitted with PEEK I.D. 0.25 mm tubing. The column was equilibrated in PCNA loading buffer supplemented with 10 mM MgCl2. Fractions were analyzed on 4–12% gradient SDS-PAGE run in MES buffer.We used pUC19 plasmid as DNA template for PCNA loading. This plasmid was nicked using the restriction enzyme Nt.BspQI for 8 h at 50°C, and was subsequently purified via phenol chloroform extraction. Reactions were performed in PCNA loading buffer 50 mM HEPES pH 7.5, 200 mM NaCl, 0.01% IGEPAL CA-360, 1mM TCEP. PCNA (30 μM) was incubated for 5 min at 30°C with nicked pUC19 (0.3 μM) and RFCΔN (0.5 μM), in the presence of MgCl2 (8 mM) and ATP (10.9 mM). This reaction was incubated at 30°C for 5 min. Then, samples were diluted with 25μl of NA buffer in order to decrease the high concentration of MgCl2 which hinder proper nucleosome assembly, followed by addition of CAF-1•H3–H4  to a final volume of 40 μl total. This tetrasome assembly step was incubated at room temperature for 15 min. Then, we added fluorescently labelled H2A–H2B dimers (0.1 μM) and incubated for 15 min at room temperature, to complete nucleosome formation (2) at 37°C for 10 min. MNase was inactivated by addition of EDTA. A 621 bp DNA fragment was added as loading control and the DNA was further purified as in  as template for PCNA loading and nucleosome assembly. pRC1765 was nicked using the restriction enzyme Nt.BbvCI for 6 h at 37°C, and was subsequently purified via phenol chloroform extraction. PCNA was loaded on DNA in PCNA loading buffer, in a final volume of 11 μl: PCNA (10.9 μM) was added to an equimolar mixture of nicked and supercoiled pRC1765 (47.3 nM each), RFCΔN (1.1 μM) in presence of MgClormation . Samplesed as in . MNase-ded as in . Data wahttps://osf.io/2vd4z/?view_only = 5ffa1e0b749445da9b22a11577f3d47f). PCNA-NAQ-seq analysis was performed using custom scripts (https://github.com/deLaatLab/PCNA-NAQ-seq). The sequence data was demultiplexed by extracting reads that contained the ligated adapter index in both read ends and trimmed by removal of the 5’ adapter sequence from the reads. Demultiplexed reads were mapped against the pLox3, pRS415 and loading control DNA sequences using BWA mem v0.7.17 and filtered using samtools with SAM flag 780 and mapping quality 60 and saved as bam files. The bam files were imported in R and fragments mapping to pLox3 and pRS415 with fragment lengths between 125 and 160 bp were selected for further analysis. The percentage of reads mapping to the nicked plasmid was calculated based on the total amount of reads found on both nicked and supercoiled plasmids. For coverage analysis pLox3 and pRS415 fragments were normalized for the total number of fragments mapping to the loading control sequence.MNase-seq was used to quantify nucleosome assembly in the PCNA-NAQ assay. In order to distinguish nucleosomes made on nicked and supercoiled DNA, we used two plasmids with different sequences: pRS415 and pLox3 . After M2, 1 mM TCEP, 1 mM ATP and 0.2 mg/ml BSA. Experiments with Polδ were performed in 25 mM Tris–HCl pH 7.5, 150 mM NaCl, 8 mM MgCl2, 1 mM TCEP, 1 mM ATP and 0.2 mg/ml BSA. We used single stranded plasmid DNA as template for DNA synthesis, and it was produced as previously described   was incu2) at 37°C for 10 min. MNase was inactivated by addition of EDTA. A 621bp DNA fragment was added as loading control and the DNA was further purified as in  were added just before MNase digestion. For MNase analysis, 30 μl of primer extension reactions at the final time point (16 min for Polϵ and 4 min for Polδ) were mixed with 80 U of MNase in a total of 100 μl , 3 μM PCNA K164C, 0.15 μM RFCΔN, 0.3 μM nicked (with Nt.BspQ1) pUC19 plasmid (from 1 μM stock), 1 mM ATP. This mixture was incubated at 30 °C for 5 min to increase the efficiency of PCNA loading onto DNA. Then, 1.5 μM CAF-1 was added and incubated for 10 min at room temperature. The total NaCl concentration during the loading reaction and after adding CAF-1, taking into account the contributions from each component, ranged between 100 and 110 mM. Samples were diluted 2-fold in buffer containing 20 mM HEPES pH 7.5, 100 mM NaCl, 1 mM TCEP, 0.02% IGEPAL CA-630, and incubated at room temperature for 10 min. Samples were subjected to chemical crosslinking by addition of a final concentration of 0.2% glutaraldehyde (from a 2.5% stock in water). The samples were incubated at room temperature for 20 min before quenching the crosslinker by addition of 100 mM Tris pH 7.5 (from 1 M stock). To release the crosslinked complexes from the DNA, 10% of the sample volume Pierce universal nuclease, diluted 1:20 in buffer containing 20 mM HEPES pH 7.5, 100 mM NaCl, 5 mM MgClLinear DNA fragments with lengths of 18, 33, 43 or 53 bp were mixed with PCNA-C4S-K164C and CAF-1 in buffer containing 20 mM HEPES–KOH pH 7.6, 100 mM KCl, 0.01% IGEPAL CA-630 and 1 mM TCEP, and incubated on ice for 10 min. The final mixture contained 1.5 μM DNA, 4.5 μM Alexa Fluor 456 labeled PCNA (concentration for a monomer), and 3 μM CAF-1. Samples were subjected to chemical crosslinking by diluting 3-fold in the same buffer and addition of a final concentration of 0.2% glutaraldehyde (from a 2.5% stock in water). The samples were incubated at room temperature for 20 min before quenching the crosslinker by addition of 100 mM Tris (from a 25× TAE stock containing 1 M Tris). Samples were spun down for 5 min at 13 000×g at 4°C and the supernatant was transferred to a new tube.Histones H3–H4  tetramers, labeled with AlexaFluor 488, were concentrated in 20 mM HEPES pH 7.5, 2 M NaCl, 1 mM EDTA, 1 mM TCEP to a final concentration of 79.4 μM using an Amicon Ultra-0.5 centrifugal concentrator with a molecular weight cut off of 10 kDa. CAF-1 WT or mutants were diluted to a concentration of 27.1 μM in 20 mM HEPES pH 7.5, 200 mM NaCl, 1 mM EDTA, 1 mM TCEP. CAF-1 was then mixed with the histones in a volumetric ratio of 3:1 to obtain samples with final concentrations of 20 μM CAF-1 and 10 μM H3–H4 tetramers. The NaCl concentration in these samples was around 650 mM. CAF-1–H3–H4 samples were mixed in order with buffer containing 20 mM HEPES pH 7.5, 60 mM NaCl, 1 mM TCEP and then PCNA-C4S-K164C  to obtain final concentrations of 1.5 μM CAF-1–H3–H4, 5.55 uM PCNA with a total of about 105 mM NaCl. Samples were diluted 2-fold in buffer containing 20 mM HEPES pH 7.5, 100 mM NaCl, 1 mM TCEP, 0.02% IGEPAL CA-630, and incubated at room temperature for 10 min. Samples were subjected to chemical crosslinking by addition of a final concentration of 0.2% glutaraldehyde (from a 2.5% stock in water). The samples were incubated at room temperature for 20 min before quenching the crosslinker by addition of 100 mM Tris pH 7.5 (from 1 M stock). Samples were spun down for 15 min at 13 000×g at 4 °C and the supernatant was transferred to a new tube.2, 1 mM ATP. Then, CAF-1 or buffer control was titrated between 0–1 μM. The total NaCl concentration during the loading reaction and after addition of CAF-1, taking into account the contributions from each component, ranged between 100 and 110 mM. After 10 min at room temperature, the samples were diluted 4.5-fold by adding buffer containing 20 mM HEPES–KOH pH 7.6, 100 mM NaCl, 0.01% IGEPAL CA-630, 1 mM TCEP, before cross-linking with 0.2% glutaraldehyde. The cross-linking reaction took place at room temperature for 20 min, after which, it was quenched with a final concentration of 100 mM Tris–HCl pH 7.5. DNA was digested using Pierce™ Universal Nuclease for Cell Lysis (Thermo Fisher Scientific) diluted to 1:20 in 20 mM HEPES–KOH pH 7.6, 100 mM NaCl, 5 mM MgCl2, 1 mM TCEP and added to 10% of the crosslinking reaction volume. The digestion was quenched with 50 mM EDTA and immediately spun down for 5 min at 13 000×g and 4°C to remove precipitates.AlexaFluor546-labeled PCNA K164C (50 nM) was loaded onto nicked (with Nt.BspQ1) pUC19 plasmids (15 nM) by RFC (15 nM). The reaction was conducted at 30 °C for 5 min in buffer containing 20 mM HEPES–KOH pH 7.6, 130 mM NaCl, 0.01% IGEPAL CA-630, 1 mM TCEP, 10 mM MgClAlexaFluor546-labeled PCNA K164C (50 nM) and FEN1_DA (0–0.6 mM) were mixed in buffer containing 20 mM HEPES–KOH pH 7.6, 100 mM NaCl, 0.01% IGEPAL CA-630, 1 mM TCEP. After 10 min at room temperature, the samples were diluted 4.5-fold by adding buffer containing 20 mM HEPES–KOH pH 7.6, 100 mM NaCl, 0.01% IGEPAL CA-630, 1 mM TCEP, before cross-linking with 0.2% glutaraldehyde. The crosslinking reaction took place at room temperature for 20 min, after which, it was quenched with a final concentration of 100 mM Tris–HCl pH 7.5. Crosslinked samples were immediately spun down for 5 min at 13 000×g and 4°C to remove precipitates.Crosslinked samples were mixed with 4× XT sample buffer and 20x XT reducing agent in appropriate volumetric ratios (#1610791 and #1610791 from Bio-Rad). These samples were loaded on 12% Criterion XT Bis–Tris gels (#3450118 Bio-Rad) in XT MOPS buffer (#1610793 Bio-Rad). Gels were run at 20 mA until the samples have completely entered the gel and then at 40 mA until the gel run was complete . Gels were run at room temperature, and additionally in the dark if components contained fluorophores. Gels were scanned for histones H3–H4 and/or PCNA fluorescence (depending on the assay) on an AMERSHAM ImageQuant 800 imager (Cytiva). Bands intensity was calculated using the ROI manager tool in Image J/Fiji and plotted using GraphPad Prism. Where applicable, gels were subsequently stained with Coomassie blue and scanned on AMERSHAM ImageQuant 800 imager (Cytiva).Samples were prepared using crosslinking at stoichiometric conditions, the reactions  were concentrated to 500 μl and loaded on a pre-equilibrated Superose 6 10/300 increase GL (Cytiva) column in 20 mM HEPES–NaOH pH 7.5, 200 mM NaCl, 1 mM TCEP. Fractions were analyzed on SDS PAGE, the ones containing the complex of interest (Peak1 or Peak2) were pooled and concentrated to about 40 μl (Abs280 close to 0.5). The samples were diluted 10 to 20-fold in 20 mM HEPES–NaOH pH 7.5, 100 mM NaCl, 1 mM TCEP right before measuring on a Refeyn OneMP instrument (Refeyn Ltd). For each measurement, 13 μl of this buffer was first placed into the CultureWell gaskets wells (Grace Biolabs) placed into the Microscope coverslips . After adjusting the focus, 2 μl of sample was mixed in. Movies were recorded for 60 seconds at 100 frames per second. A calibration measurement under the same conditions was performed roughly every 15 measurements using an in-house prepared protein standard mixture: IgG4Δhinge-L368A (73 kDa), IgG1-Campath (149 kDa), apoferritin (479 kDa), and GroEL (800 kDa). Data were processed using DiscoverMP (Refeyn Ltd) with bin width adjusted to 10, and each sample retrieved about 1500–3000 counts. Figures were prepared with the Refeyn instrument and edited in Illustrator.Kd values were calculated using a one site binding curve in GraphPad Prism. Representative curves are shown from one experiment (three independent measurements) and were repeated at least two times in triplicates.Fluorescence Polarization assays were carried out in 25 mM TRIS pH 7.5, 300 mM NaCl, 5% glycerol, 1 mM EDTA, 0.01% IGEPAL ca-630 (added fresh), 0.01% CHAPS (added fresh), 1 mM DTT (added fresh). Binding reactions were prepared by mixing 10 nM of Alexa488-labeled H3–H4 (H3 C110A-H4 E63C) and increasing amounts of CAF-1, Polϵ, Polδ or RPA in a final volume of 30 μl in CORNING low flange 384 well black microplates (CLS3575). Binding data were measured using a CLARIOStar (BMG LabTech) plate reader. The data was analyzed and plotted using Microsoft Excel and GraphPad Prism. This refers to 2. Media was prepared by supplying DMEM-GlutaMAX-pyruvate with fetal bovine serum (15%), LIF (made in house), 1x non-essential amino acids (Gibco), 1× penicillin/streptomycin (Gibco) and 2-beta-ME (0.1 μM). Cells were passaged using Trypsin-EDTA (Gibco) or TrypLE (Gibco). Cells were routinely tested for mycoplasma contamination. For genome editing Chaf1a-dTAG cells were generated by CRISPR-Cas9 using the SpCas9(BB)-2A-Puro (PX459) V2.0 plasmid (addgene #62988) as described in  in serum + LIF conditions at 37 °C with 5% COribed in  with sgRribed in , which tribed in . Positivribed in  and degrribed in . Fractioribed in . Westernribed in .Cells treated with DMSO or dTAG-13 for 4 h, were pulsed in EdU-containing media (10 μM) for 10 min and immediately fixed for 15 min in 4% PFA at room temperature and stored in PBST (PBS with 0.3% Triton X-100). Primary antibody H4K20me0 was added at the concentration of 1:1000 in PBST with 5% donkey serum and incubated overnight. Incubation was followed by three washes in PBS and secondary antibody was then added in PBST. Samples were incubated with the secondary antibody in the dark at room temperature for 1 h. After three washes, samples were stained with DAPI (1:10 000) in PBST. Images were acquired with a ScanR high-content screening microscope (Olympus). Automated and unbiased image analysis was carried out with the ScanR analysis software (version 2.8.1). Individual cells were identified based on DAPI staining and mean pixel intensity was measured for each channel. Data were exported and processed using Spotfire software . Statistical analysis and visualization of results was done using using R (v4.1.2) in RStudio (v2021.9.2.382).Chaf1a-dTAG cells in three biological replicates for each histone PTM. Cells treated with DMSO or dTAG-13 for 2 h, were pulsed in EdU-containing media (10 μM) for 30 min and harvested immediately. For sample collection, media was aspirated, plates washed 2× with room temperature PBS and ice-cold PBS was added to the dishes. Cells were scraped in a cold room and collected by centrifugation, followed by nuclei isolation. Nuclei were aliquoted, snap-frozen and stored at -80°C until further use. For MNase digest, nuclei were counted manually using Kova Glasstic Slides and 2 U MNase (Worthington) were added per 1 × 106 nuclei. Digests were performed at 30°C for 20 min. For native ChIP, 30–50 μg of chromatin was used per sample and incubated with antibodies in a total volume of 600 μl overnight at 4°C with H3K27me3 antibody  or H4K20me0 antibody . Magnetic beads  were added the next morning and samples were incubated for 2 h. After three washes each with ice-cold RIPA buffer and RIPA 0.5M NaCl buffer, DNA was eluted and purified using the MinElute Reaction Cleanup kit (Qiagen). Mononucleosomal-sized fragments were isolated by double sided size selection with AMPure XP beads (Beckman Coulter). EdU-labelled DNA fragments were biotinylated using Click-iT chemistry as reported above but using Biotin-TEG-Azide (Berry & Associates) instead of Picolyl-azide-PEG4-Biotin. Libraries were prepared using the KAPA Hyper Prep Kit (Roche). Biotinylated fragments were captured using Dynabeads MyOne Streptavidin (invitrogen) and EdU strands were purified by performing NaOH washes. Libraries were amplified in 9–11 PCR cycles. Libraries with mononucleosomal-sized inserts were isolated by double-sided size selection with AMPure XP beads (Beckman Coulter), followed by a second clean-up with 1.0x AMPure XP beads. Fragment distribution of libraries was checked on a Fragment Analyzer system (Agilent). Stranded input samples were prepared in parallel with SCAR-seq samples. Samples were sequenced single end (75bp) on a NextSeq500 instrument (Illumina).A step-by-step protocol is available . BrieflyReads were processed, mapped and histone partition signal was computed as described previously 68). Briefly, for each strand the SCAR normalized signal (CPM) was computed in 1kb bins and smoothed in a uniform blur considering the neighbouring 30 bins on each side. For each 1kb window, the signal from its corresponding SCAR input was subtracted and negative values were set to zero. Input corrected windows with CPM 0.3 on both strands were filtered out and not considered for further analyses. The final partition score for each 1kb window was calculated as: partition = (F – R)/(F + R) where F and R correspond to the number of normalized and input-corrected reads for the forward and reverse strand, respectively. The partition value relates to the ratio of histones with a specific modification being segregated to the nascent forward (partition > 0) or nascent reverse (partition < 0) strand within each window respectively. Okazaki-seq replication fork directionality (RFD) scores and filtered initiation zones (IZs) for mESC were taken from , was used as the max size of replicated products  region located at the N-terminus, which is flanked by the PIPs, and the winged-helix domain (WHD) at the C-terminus . Either  of PCNA . We thus of PCNA . However of PCNA . These rKd > 2 μM) than to a 33, 43 or 53 bp DNA   , in linectively) . Mutatioctively) . Complexctively) . This suctively)  and corrctively) . This doin vitro in the absence of other factors -based analysis of nucleosome fragments (±150 bp) are also used to assess nucleosome formation. We named this setup PCNA-NAQ assay, based on our previously established Nucleosome Assembly and Quantitation (NAQ) assay  activitQ) assay ,63.bona fide measure of assembled nucleosomes. Quantification of the histone fluorescence signal on the nicked plasmid compared to total histone signal showed that roughly 50% of the nucleosomes are assembled in a PCNA-dependent manner, when PCNA is loaded  on the nicked plasmid was observed only when PCNA is loaded on DNA and CAF-1 is present Figure . When PCd Figure . Thus, win vitro reconstitutions that omitted PCNA were unable to recapitulate loss of function of this mutant  than Polϵ   (Xenopus laevis FEN1 D181A (FEN1_DA), which binds yeast PCNA as a monomer in a PIP-dependent manner and with affinities that are comparable to CAF-1 (Previous studies have shown that Polδ has a higher binding affinity for PCNA ( 326 nM) . We foun 326 nM) , D. Theras Polδ) . While ias Polδ) . This suto CAF-1  80). FE. FEKd,apto CAF-1 . This inDuring DNA replication in cells, Polϵ and CAF-1 both bind H3–H4 ,82–83. TKd of 28 nM. This is a 25 times lower affinity than that of CAF-1 (Kd = 1.1 nM) (Kd ≥ 300 nM), similarly to RPA which is also present in the reactions (Kd ≥ 300 nM)  . Neverth 300 nM) . These dTo test the effect of histone binding in the crosstalk between CAF-1 and the DNA polymerases, we pre-incubated either the DNA polymerase or CAF-1 with H3–H4 and monitored how this affected DNA synthesis in primer extension assays. Polϵ activity was not affected by the addition of H3–H4 Figure  and the Previous studies have shown that CAF-1, Polϵ and RPA can assemble chromatin during DNA replication ,82–84. AWe identified a differential crosstalk of CAF-1 with Polϵ and Polδ, likely through their differential interaction with PCNA. As CAF-1 and Polϵ compete for binding on PCNA, we wondered whether CAF-1 is able to assemble nucleosomes on the leading strand. To address this question directly in cells, we used mouse embryonic stem cells (mESCs) and employed Sister Chromatid after Replication Sequencing (SCAR-seq) ,69. ThisChaf1a). dTAG-Chaf1a is targeted for proteasomal degradation in the presence of the degrader compound dTAG .To focus on Polϵ activity, we used a pulse-chase setup in which we omitted Polδ. This allowed us to quantify replication rates of the leading strand only , by moniTogether, these data, combined with the observation that CAF-1 acts on both daughter strands Figure , supportOur work provides insights into how the essential histone chaperone CAF-1 functions during genome replication. We show that CAF-1 recruitment and its PCNA-dependent nucleosome assembly activity are regulated by a complex set of interactions between CAF-1 and PCNA, DNA and histones. Our results argue that several structural transitions regulate CAF-1, and we anticipate that these control the timing of CAF-1 arrival to, action on and departure from replication forks. This is linked with the interplay between CAF-1, PCNA and the DNA polymerases. CAF-1 competes with Polϵ for PCNA binding, while it has no effect on Polδ. Yet, CAF-1 deposits newly synthesized histones equally on both strands in mESCs. Therefore, the competition between CAF-1 and Polϵ appears to be an integrated part of coordinating replication and nucleosome assembly and does not limit CAF-1 function. Our work suggests that different mechanisms are in place on the leading and lagging strand to couple DNA synthesis with CAF-1 mediated nucleosome assembly, in line with the inherent asymmetry of DNA replication and its chromatin assembly mechanisms.in vivo. Both models imply the need for regulatory steps in PCNA accessibility and loading on the leading strand, where the CTF18 clamp loader that binds Polϵ may well play a role .,75.54,75Fastaq files from MNase-seq are uploaded to OSF and can be accessed with the following link:https://osf.io/2vd4z/?view_only=5ffa1e0b749445da9b22a11577f3d47fhttps://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE223782)SCAR-seq data are available with GEO accession GSE223782 (gkad171_Supplemental_FileClick here for additional data file.


Through the combination of the chemical and photophysical properties of Ce6 and C60, in principle, we can obtain an “ideal” photosensitizer that is able to bypass the limitations of the two molecules alone, i.e., the low cellular uptake of Ce6 and the scarce solubility and absorption in the red region of the C60. Here, we synthesized and characterized a Ce6–C60 dyad. The UV-Vis spectrum of the dyad showed the typical absorption bands of both fullerene and Ce6, while a quenching of Ce6 fluorescence was observed. This behavior is typical in the formation of a fullerene–antenna system and is due to the intramolecular energy, or electron transfer from the antenna (Ce6) to the fullerene. Consequently, the Ce6–C60 dyad showed an enhancement in the generation of reactive oxygen species (ROS). Flow cytometry measurements demonstrated how the uptake of the Ce6 was strongly improved by the conjugation with C60. The Ce6–C60 dyad exhibited in A431 cancer cells low dark toxicity and a higher PDT efficacy than Ce6 alone, due to the enhancement of the uptake and the improvement of ROS generation.Chlorin e6 (Ce6) and fullerene (C An ideal photosensitizer ,2 must h60) + max 430 nm, 327 nm, 257 nm.UV-Vis (DCM)  λmax 4304  in DCM (1 mL) and trifluoroacetic acid (1 mL) was stirred at room temperature for 3 h (60∙2TFA) and as a brown solid  in a 94% yield. A solution of  for 3 h . The sol+ 806.98.ESI-MS (MeOH) : [M+H]+ max 433 nm, 325 nm.UV-Vis (DMSO) : λmax 435  and TEA  in anhydrous DCM (10 mL) , EDC∙HCl , and HOBt  were dissolved in anhydrous DCM (5 mL) and stirred at room temperature for 1 h. This solution was added dropwise to a suspension of the fullerene derivative  (10 mL) . The res− 1383.3271, [M-CO2]− 1339.3300, [M+OH]− 1400.3185, [M+CH3O]− 1414.3060.HR ESI-MS (MeOH) : [M-H]− max 672 nm, 615 nm, 539 nm, 506 nm, 409 nm, 322 nm, 255 nm.UV-Vis (DCM): λ1H NMR : δ 9.63 , 9.51 , 8.61 , 7.97 , 6.26 , 6.14 , 4.44 , 4.15–4.05 , 4.04–3.93 , 3.80 , 3.36 , 3.26 , 3.19 , 2.62–2.52 , 2.35 , 1.76–1.62 .ABMDMA assay. 9,10-Anthracenediyl-bis(methylene)dimalonic acid (ABMDMA) was used as singlet oxygen detector. The disodium salt of ABMDMA reacts with 1O2 to give an endoperoxide  in order to test the final concentrations of 5, 1, and 0.5 μM. Another well was filled with only deuterated PBS and DMSO (5%). Lastly, 3 μL of an ABMDMA 5 mM stock solution in DMSO was added to each well. The plate was irradiated for 1 h with a red light lamp.DMSO stock solutions of Ce6 and Ce6–CΔ) of Ce6 and Ce6–C60 was determined by irradiating the samples with the red lamp for up to 20 min and following the ABMDMA bleaching over time, with timesteps of 5 min. The ΦΔ was calculated using the following equation:k is the slope of the photooxidation rate of ABMDMA obtained by the curve-fitting equation .The singlet oxygen quantum yield (Φequation . AbsorbaAmplex Red assay. The Amplex Red (AR) assay was used to evaluate the generation of peroxides upon irradiation with visible light. Colorless and nonfluorescent Amplex Red reacts with peroxides in a reaction catalyzed by horseradish peroxidase (HRP) to form Resorufin, which is a fluorescent molecule  to obtain a solution characterized by a final AR concentration of 500 μM. The final working solution (WS) was obtained by adding 10 μL of HRP at 0.4 mg/mL in PB to the AR solution. Stock solutions of Ce6 and Ce6–C60 in DMSO were prepared, with concentrations of 100, 20, and 10 μM. In a 96-well plate, 5 μL of these solutions was added to 85 μL of PB . The plate was irradiated with a red light lamp for 1 h and then 10 μL of the WS was added to each well. An equivalent plate was kept in the dark and then the WS was added. After 30 min, the fluorescence data were collected using a PerkinElmer EnSpire® Multimode Plate Reader .The concentration of the produced peroxides was calculated by measuring the fluorescence emission of Resorufin at 590 nm (excitation at 560 nm), subtracting the values of non-irradiated references that were kept in the dark. The fluorescence values were converted into hydrogen peroxide concentrations using a calibration curve obtained from standard solutions of HIntracellular ROS Production. Intracellular ROS production after PDT was determined by using ROS-Glo™ H2O2 Assay , following the protocol provided by the manufacturer. Briefly, around 25,000 A431 cells (ATCC CRL-1555) were incubated for 3 h with increasing concentrations of either Ce6 or Ce6–C60 prior to washing and irradiation with a red light lamp for 45 min. Immediately after the treatment, 20 µL of H2O2 substrate solution was added to each well  and incubated for 20 min at 37 °C. A total of 100 µL of ROS-Glo™ detection solution was added to each sample, and the plate was incubated at room temperature for 20′. Relative luminescence was measured using EnSpire® multimode plate reader .60 and Ce6 was evaluated on an A431 human epidermal carcinoma cell line (ATCC CRL-1555). A total of 100,000 cells were seeded in a 6-well plate and incubated overnight at 37 °C with 5% CO2. Next, cells were incubated with either 0.1 μM or 0.03 μM concentrations of Ce6–C60 or Ce6 for 20, 60, or 180 min. After the incubation, cells were washed twice with PBS to remove any unbound sensitizers. Subsequently, the cells were detached by trypsinization and analyzed using a citoFLEX S flow cytometer . Data analysis was performed using CytExpert software 2.5 (Beckman Coulter) and FlowJo™ 10.9 software.Cellular uptake of Ce6–CMTT assay. In a 96-well flat-bottom plate , 25,000 cells of confluent growing A431 cells were seeded in 100 µL complete growth media RPMI  per well and incubated overnight at 37 °C with 5% CO2. Cells were then incubated with increasing concentrations of Ce6 or Ce6–C60 (diluted in RPMI) for 3 h at 37 °C with 5% CO2. Afterwards, cells were washed twice with 1× PBS to remove any free sensitizer. For irradiation, 100 µL of 1× PBS was added per well, and the 96-well plate was irradiated for 45 min with a red lamp  for 1 min per well.2. The dark control plate was treated the same way but kept in the dark throughout. The cell viability was tested using MTT assay. Therefore, the cells were incubated in a final concentration of 0.5 mg/mL MTT  in 100 µL RPMI per well for 90 min at 37 °C with 5% CO2. Next, the media were removed, and 100 µL per well of DMSO was added for the formazan solubilization. To determine the cell viability, the absorbance was measured at 570 and 690 nm wavelengths using the EnSpire multimode plate reader , and the absorbance at 570 nm was subtracted from the absorbance at 690 nm. The obtained values were normalized against a growth control that was not treated with sensitizer. The results are shown as the mean ± SD of three biological replicates, each with three technical replicates.Cells were allowed to recover overnight in 200 µL complete RPMI per well at 37 °C with 5% COPropidium iodide staining. A431 cells were seeded on round coverslips placed inside a 6-well plate (Corning) and grown overnight at 37 °C with 5% CO2. Cells were then incubated in MTT assay, as described above, with either Ce6 or Ce6–C60 at a final Ce6 equimolar concentration of 0.1 μM and (i) kept in dark conditions, (ii) irradiated for 45 min with a red lamp (6.3 mW/cm2), or (iii) irradiated for 1 min with laser at 660 nm (2.66 W/cm2). Cells were then recovered for 24 h and stained for 30 min with Propidium iodide (PI) at a final concentration of 1 μM. A round coverslip was fitted into an Attofluor cell chamber  and covered with 1 mL of RPMI without phenol red complete . Cell images were then acquired with a Nikon Eclipse Ti-U inverted epifluorescence microscope.60 dyad. The Ce6–C60 dyad was synthesized via a coupling reaction between the carboxylic groups of the Ce6 molecule and a C60 derivative bearing an amine group.In summary, we developed a novel phototheranostic platform for PDT treatment/fluorescence imaging based on the Ce6–C60, the obtained Ce6–C60 dyad had the following characteristics: (i) efficient absorption in the red region of the visible spectrum; (ii) production of ROS following both type I and type II photophysical mechanisms; (iii) good biocompatibility and solubility in physiological environments; (iv) enhanced cellular uptake efficiency in vitro (with a fluorescence increase of 10.5 times after 3 h of incubation in comparison to Ce6); (v) simultaneous imaging capability and PDT performances. Remarkably, the dyad displayed superior photo-capabilities in terms of both peroxide generation and PDT efficiency on cancer cells. Notably, dyad irradiation resulted in the production of five times more peroxides and the photokilling of 40.2% more cancer cells compared to Ce6 at the same equimolar concentration.Combining the peculiar properties of Ce6 and C60 dyad suggest that, in perspective, it can be used as a new red light phototheranostic agent for fluorescence-imaging-guided PDT treatment.This approach paves the way for the molecular engineering of highly efficient fullerene-based photosensitizers for anticancer PDT. The unique properties of the Ce6–C


The bmp8a-/- zebrafish exhibits decreased phosphorylation levels of AMPK and ACC in the liver and adipose tissues, indicating reduced fatty acid oxidation. Also, Bmp8a inhibits the differentiation of 3T3-L1 preadipocytes into mature adipocytes by activating the Smad2/3 signaling pathway, in which Smad2/3 binds to the central adipogenic factor PPARγ promoter to inhibit its transcription. In addition, lentivirus-mediated overexpression of Bmp8a in 3T3-L1 cells significantly increases NOD-like receptor, TNF, and NF-κB signaling pathways. Furthermore, NF-κB interacts with PPARγ, blocking PPARγ’s activation of its target gene Fabp4, thereby inhibiting adipocyte differentiation. These data bring a signal bridge between immune regulation and adipocyte differentiation. Collectively, our findings indicate that Bmp8a plays a critical role in regulating lipid metabolism and adipogenesis, potentially providing a therapeutic approach for obesity and its comorbidities.The role of bone morphogenetic proteins (BMPs) in regulating adipose has recently become a field of interest. However, the underlying mechanism of this effect has not been elucidated. Here we show that the anti-fat effect of Bmp8a is mediated by promoting fatty acid oxidation and inhibiting adipocyte differentiation. Knocking out the  Bone morphogenetic protein 8a (Bmp8a) is required for fatty acid oxidation and regulates adipogenesis as evidenced by weight gain and fatty liver of zebrafish lacking Bmp8a. Generally, obesity is characterized by a massive expansion of white adipose tissue due to an increase in the size or number of adipocytes and a decrease in lipolysis. Therefore, identifying the factors that regulate adipose tissue expansion and elucidating the mechanisms are vital for public health and will help formulate therapeutic strategies and targets for the treatment of obesity and its associated comorbidities.Obesity and overweight have become a worldwide epidemic. Obesity is strongly associated with many metabolic and cardiovascular diseases, such as type 2 diabetes, dyslipidemia, hypertension, some types of cancer, and osteoarthritis7, and bone morphogenetic proteins (BMPs) are a relatively recent addition to the adipose regulation field. BMP belongs to the transforming growth factor-β (TGF-β) superfamily, which is highly conserved in developing vertebrates ranging from humans to zebrafish9. They were initially discovered as inducers of bone and cartilage10, but have been known to be critical in morphogenetic activities and cell differentiation throughout the body, including the development of adipose tissue and adipogenic differentiation15. BMP2, BMP4, and BMP6 have been shown to promote white adipogenesis in mesenchymal stem cells18. BMP7 and BMP8B can induce the expression of UCP1, a marker gene of brown adipocyte, and promote brown adipogenesis or enhance the thermogenesis of brown adipose tissue23. In addition, BMP3B suppresses adipogenesis of 3T3-L1 cells25. Overexpression of BMP9 in the mouse liver significantly alleviates hepatic steatosis and obesity-related metabolic syndrome26.Adipogenesis is regulated by a variety of signaling pathways23. Our previous studies have shown that Bmp8a can accelerate the uptake of yolk sac in zebrafish at 3-dpf (days post fertilization), indicating that Bmp8a may play a key role in fat metabolism27. Nevertheless, the overall metabolic regulatory function of BMP8A is not fully understood. Here, we report that bmp8a-/- zebrafish display obesity and fatty liver. Deletion of bmp8a in zebrafish leads to the accumulation of liver TG by downregulating phosphorylation of AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC). Furthermore, Bmp8a inhibits the differentiation of 3T3-L1 preadipocytes into mature adipocytes through the Smad2/3 pathway. Interestingly, we also found that the interaction of NF-κB and PPARγ mediates the effect of Bmp8a on adipogenesis, providing a signaling bridge between immunomodulatory and adipocyte differentiation. We present a previously unrecognized insight into Bmp8a-mediated adipogenesis.BMP8A is almost absent from brown adipose tissue, whereas it is enriched in white adipose tissue27. Here the bmp8a-/- zebrafish was used to perform further analysis28. We monitored the body weight of wild type (WT) and bmp8a-/- zebrafish fed with a high-fat diet (HFD) and found that the body weight of bmp8a-/- zebrafish was gradually higher than WT zebrafish   Fig. . Oil Redish Fig. , which wish Fig. . Zebrafivae Fig. . Importates Fig. . Taken tbmp8a-/- zebrafish had higher levels of blood GLU, TG, and TC than WT zebrafish  technology. It was shown that in the liver or adipose tissue of bmp8a-/- zebrafish fed a high-fat diet, genes for lipolytic enzymes (lpl and lipc), insulin-sensitizing hormone (adiponectin), transcription activators and coactivators that induce fat metabolism (pgc-1α and pparα), mitochondrial proteins that used to generate heat by thermogenesis (ucp1), and hunger suppressors (leptin) were all downregulated , was upregulated in the tissue of the liver or intestine of HFD-fed bmp8a-/- zebrafish  during 3T3-L1 differentiation , followed by insulin treatment (Day 2 ~ Day 4), these cells can be induced to differentiate into adipocyte-like cells. Intracellular lipid accumulation can be observed and quantified after staining with Oil Red O, providing an effective model system for adipogenesis in vitro Fig. . Firstlyion Fig. . Obviousion Fig. . Then, wlls Fig. . We founion Fig. . In contp8a Fig. . Also, oasn Fig. . Proteinlls Fig. . Furtherasn Fig. . Consistsed Fig. . These r34. We evaluated the activation of these signals by immunoblot assays in both Bmp8a-overexpressing and WT 3T3-L1 cells. Phosphorylation levels of Smad1/5/8 and Smad2/3, but not ERK, JNK, or p38 MAPK, were significantly increased in zebrafish bmp8a or mouse Bmp8a-overexpressing 3T3-L1 cells  and Smad-independent pathways lls Fig. . Then, tlls Fig. . Thus, i36. Interestingly, the type I receptor Alk6 gene was not found in the 3T3-L1 cells  and type II receptors  during adipocyte differentiation were initially elevated and then progressively decreased. Also, the expression of the Alk7 gene increased gradually alongwith adipocyte differentiation. To further examine the signal transduction pathway mediated by Bmp8a, BRE- and CAGA-driven luciferase reporter assays were performed. In this system, the BRE promoter was activated by signaling through Smad1/5/8, while the CAGA promoter was activated by signaling through Smad2/337. Bmp8a could activate Smad1/5/8 signaling through receptor complexes formed by the type I receptor ALK3 and the type II receptor BMPR2 or ACVR2A  and two type II receptors  Fig. 35,36. Inlls Fig. . Next, wnes Fig. . In paranes Fig. . The expR2A Fig. . Also, BR2A Fig. . NotablyAlk3-ΔGS, Alk4-ΔGS, and Alk5-ΔGS) plasmid  to search for potential Smad2/3 transcription factor (TF) binding sites in the Pparγ promoter. There are three potential TF binding sites with high scores according to the prediction   in the Pparγ promoter attenuated the ability of Bmp8a to suppress Pparγ promoter activity, suggesting that Region 1 may be the region where Smad2/3 binds to the Pparγ promoter  Fig. . We founter Fig. .bmp8a, LV-Bmp8a, and LV-ZsGreen1 3T3-L1 cells. To identify the differentially expressed genes (DEGs) in the two cell types (LV-bmp8a or LV-Bmp8a), the cutoff values for the fold change and P value were set to 2.0 and 0.05, respectively. Among the DEGs, 2337 genes (1215 downregulated genes and 1122 upregulated genes) were significantly modulated in LV-bmp8a cells, while 2187 genes (1211 downregulated genes and 976 upregulated genes) were modulated in LV-Bmp8a cells. By combining the two data sets, we found the two cell types shared 536 overlapping downregulated DEGs and 334 overlapping upregulated DEGs  analyses revealed that compared with LV-ZsGreen1 3T3-L1 cells, the downregulated genes were remarkably enriched in the PPAR signaling pathway whether in LV-lls Fig. . More inlls Fig. . Given tlls Fig. .Fig. 7ThPparγ, so we want to understand the relationship between NF-κB and PPARγ in regulating adipogenesis. Previous studies have revealed that NF-κB components p50 and p65 bind PPARγ directly in vitro by GST Pull-down assay38. Our co-immunoprecipitation (Co-IP) experiments confirmed that NF-κB (p65) interacts with PPARγ , as a target gene of PPARγ, promotes adipocyte differentiation40. We found that a putative PPRE is present in the promoter region of Fabp4 , increasing the response to noradrenaline through enhanced p38MAPK/CREB signaling and increased lipase activity. Bmp8b-/- mice exhibit impaired thermogenesis and reduced metabolic rate, causing weight gain23. BMP8A is almost absent from BAT, but enriched in white adipose tissue (WAT)23. There have been no reports on the role of BMP8A in adipose tissue. In this study, we showed that zebrafish Bmp8a or mouse BMP8A has an anti-fat effect by promoting fatty acid oxidation and reducing adipocyte differentiation.The BMPs are members of a large highly conserved family of extracellular polypeptide signaling molecules of the TGF-β superfamily. In 44. We have previously shown that bmp8a mRNA expression in the intestine or brain significantly upregulated in obese zebrafish induced by a high fat diet27. In this study, we found that compared to WT, bmp8a-/- zebrafish exhibited higher body weight and increased fat production, confirming the linking of Bmp8a with obesity. Furthermore, we showed that the expression of key genes associated with lipid metabolism , thermogenesis (ucp1), and appetite (leptin) were regulated by Bmp8a. Thus, Bmp8a appears to regulate obesity through multiple molecular pathways. Overall, we speculate that Bmp8a increases lipase activities such as Lpl and Lipc to hydrolyze triglyceride (TG) into free fatty acids (FFA). Subsequently, Bmp8a enhances FFA oxidation through AMPK or p38 MAPK pathway, thereby reducing lipid accumulation 47. However, we are unsure about the causal relationship between the fatty liver and dysregulation of lipid metabolism in bmp8a-/- zebrafish. Most likely, these two aspects lead and influence each other.We also found fatty liver in 48. Adipogenic factors C/EBPα and PPARγ are two central players in white adipocyte differentiation49. Here, we illustrated the inhibitory regulation of Bmp8a on adipogenesis by decreasing the expression of adipogenic markers (C/EBPα and PPARγ). Also, the expression of pparγ was increased in both liver and adipose tissue of bmp8a-/- zebrafish compared to wild-type zebrafish  were established from the zebrafish AB line using TALENs technology28. Embryos from natural matings were grown at 28 °C. Female zebrafish were used for the experiments unless otherwise specified.All animal experiments were performed in accordance with the Institutional Animal Care and Use Committee of the Ocean University of China (SD2007695). The zebrafish 2. The ZFL cells were maintained in DMEM/F-12 media supplemented with 10% FBS and penicillin/ streptomycin at 37 °C in a humidified incubator with 5% CO2. All cell lines were subjected to the examination of mycoplasma contamination and were cultured for no more than 1 month. The cell morphology was confirmed periodically to avoid cross-contamination or misuse of cell lines.Mouse preadipocytes 3T3-L1 and human HEK293T cells were obtained from ATCC. The zebrafish liver cells (ZFL) were acquired from the China Zebrafish Resource Center (CZRC). 3T3-L1 and HEK293T cells were maintained in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum  and penicillin/streptomycin at 37 °C in a humidified incubator with 5% COBriefly, two days after reaching 100% confluency (Day 0), 3T3-L1 preadipocytes were treated with MDI cocktail, including 1 μg/ml insulin , 0.25 μM dexamethasone , and 0.5 mM isobutylmethylxanthine  for 48 h. Then, cells were transferred to DMEM with insulin (1 μg/ml) and 10% FBS for 2 days. Afterward, cells were cultured in DMEM containing 10% FBS and the medium was replaced every 2 days until more than 90% of cells showed adipocyte morphology.HEK-293T cells were co-transfected with Lentiviral vectors  and packaging vectors  using FuGENE HD Transfection Reagent  were packaged in HEK293T cells and concentrated using the Lentivirus Concentration Solution Kit . After transfection into 3T3-L1 cells, 2 μg/ml puromycin  was used to select stable cells. The survival cells were expanded and confirmed by western blot.bmp8a, mouse  were cloned into the pCMV-C-Flag vector for the eukaryotic expression, respectively. The mouse Bmp8a and p65 ORF were cloned into the pCMV-C-HA vector for the eukaryotic expression, respectively. The zebrafish bmp8a and mouse Bmp8a ORF were cloned to pLVX-mCMV-ZsGreen1-Puro vector, respectively. Dominant negative mutant plasmids Alk3-ΔGS (delete the sequence of 5’-AGCTCTGGGAGTGGATCTGGA-3’ encoding the GS domain), Alk4-ΔGS (delete the sequence of 5’-TCAGGGTCTGGCTCAGGG-3’ encoding the GS domain), Alk5-ΔGS (delete the sequence of 5’-TCAGGGTCTGGATCAGGT-3 ’ encoding the GS domain) were cloned to pLVX-mCMV-ZsGreen1-Puro vector, respectively. The sequences  were cloned to pLVX-shRNA2-Puro vector, respectively. The deletions and mutations were created using Mut Express II Fast Mutagenesis Kit V2 . All constructs were confirmed by DNA sequencing. The primer sequences used in this study were listed in Supplemental Table The open reading frames (ORF) of zebrafish Weight (g) was measured by putting the fish into a small beaker of facility water on a scale and subtracting the non-fish weight.58.Tricaine  was used at 0.02% in water, at 28.5 °C. Fish were transferred to a beaker containing Tricaine and then monitored for signs that they had reached loss of reactivity. For anesthetics, loss of reactivity was typically reached within 60 s59. In brief, fresh livers were washed two times with PBS to remove impurities such as blood and then fixed in 4% (w/v) paraformaldehyde  for 2 h at 4 °C. Tissue blocks were embedded in opti-mum cutting temperature compound . The 5 µm thick liver sections were prepared using Cryostat Microtome . Finally, samples were subjected to Oil Red O  staining. The histopathological examination of the liver was performed under a Zeiss Axio Imager A1 microscope.The tissue was fixed, dehydrated, embedded and Oil red stained according to proceduresg, for 12 min. The whole blood levels of TG, TC, and GLU were measured by a fully automated biochemistry analyzer .The zebrafish were fasted for 12 h and then anesthetized. Blood was collected by holding a heparinized microcapillary tube  after decapitation. The blood was allowed to settle for 1.5 h and then centrifuged at 4 °C, 12,000 × g for 10 min at 4 °C, and the supernatant placed on ice to be tested. The levels of TG and TC were analyzed using assay kits  purchased from Elabscience Biotechnology according to the manufacturer’s instructions.The zebrafish were fasted for 12 h and then anesthetized. Fresh liver and adipose tissues were collected, rinsed with PBS (pH 7.4) at 4 °C, blotted to filter paper, weighed, placed into a homogenization vessel, homogenized by adding Isopropanol at a ratio of weight (g): Volume (ml) = 1:9 at 4 °C, centrifuged at 10,000 × g, 4 °C, supernatants were incubated with Protein A + G Agarose  coupled to indicated antibody for overnight. The sepharose beads were washed three times with 1 ml NP-40 buffer. For immunoblot analysis, immunoprecipitates or whole-cell lysates were separated by SDS-PAGE, electro-transferred to PVDF membranes and blocked for 4 h with 4% bovine serum albumin (BSA) in PBS-T , followed by blotting with the appropriate antibodies and detection by Omni-ECL™Femto Light Chemiluminescence Kit . The membrane was visualized using a fluorescent Western blot imaging system . The integrated absorbance  of the protein bands was measured using ImageJ. The target protein expression level was presented as the ratio of the IA of the target protein to the IA of β-actin or total protein.Cultured cells were lysed in NP-40 buffer  for 20 min on ice. After centrifugation for 10 min at 13,000 × Antibodies from Affinity Biosciences: p-Smad1/5/8 , Smad1/5/8 , PPARγ , UCP1 . Antibodies from Beyotime: HA tag , Flag tag . Antibodies from Sangon Biotech: p-ACC , ACC . Antibodies from Abcam: BMP8A . Antibodies from ZenBio: p-AMPK , AMPK , PGC-1α . Antibodies from CWBIO: goat anti-Rabbit IgG HRP secondary antibody , goat anti-Mouse IgG HRP Conjugated . Antibodies from Bioss: p38 MAPK , p-p38 MAPK , β-actin , p-JNK , JNK , C/EBPα , p-Smad2/3 , Smad2/3 , p-ERK1/2 , ERK1/2 , p-p65 , p65 , p-IKKα/β , IKKα/β .Smad1/5/8 inhibitor DMH1, Smad2/3 inhibitor TP0427736 HCl , were dissolved in dimethyl sulfoxide (DMSO). The cells were treated with each inhibitor, which was diluted with culture medium at concentrations of 5 μM.Nile Red  was dissolved in acetone at 1.25 mg/ml and stored in the dark at 20 °C. Vessels containing live unanesthetized zebrafish were supplemented with Nile Red to a final working concentration of 0.5 μg/ml and then placed in the dark for 30 min. Zebrafish were anesthetized in Tricaine , mounted in 3% methylcellulose, and imaged using a Leica MZ16F fluorescence stereomicroscope.4 cell/well in 24-well plates and cotransfected with various constructs at a ratio of 10:10:1 (BRE- or CAGA-driven luciferase reporter/expression plasmid/pRL-TK) using FuGENE HD Transfection Reagent. The luciferase reporter activity was measured using the Spark 20 M multifunctional microplate reader. Data were normalized by calculating the ratio of Firefly/Renilla luciferase activity.Luciferase activity levels were measured according to the manufacturer’s instructions . Briefly, HEK293T cells were plated at 6 ×102O for 2 min and then stained in freshly diluted Oil Red O solution  was diluted with water (3:2), filtered through a 0.45 µm filter) for 30 min. Cells were then washed with 60% isopropanol in H2O and twice with PBS. The cells were observed in H2O under a microscope and photographed. Oil Red O was extracted with 100% isopropanol, and the absorbance reading was performed at OD492 nm.After induction of adipogenic differentiation, 3T3-L1 cells were rinsed three times with PBS and then fixed for 20 min with 4% PFA. The cells were treated with 60% isopropanol in Hβ-actin or mouse Gapdh). All qRT-PCR experiments were performed in triplicate and repeated three times. The primer sequences were described in Supplementary Table RNA was isolated using the Trizol Reagent . Generally, RNA was reversed to cDNAs by PrimeScript™ RT reagent Kit with gDNA Eraser . Samples without reverse transcriptase were also added as control. Gene expression was determined by amplifying the cDNA with ChamQ SYBR Color qPCR Master Mix  by using an ABI 7500 Fast Real-Time PCR System . Gene expression levels were normalized to an internal control gene (zebrafish bmp8a), and , were collected. Sequencing libraries were generated using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina . Sequencing was performed on an Illumina NovaSeq 6000, and 150-nucleotide paired-end reads were generated. At least 6 GB of clean data with >94% of them above Q30 were produced for each sample. HISAT2 and StringTie were used to align the reads and to analyze the transcripts61. The DEGseq R package was used to identify differentially expressed genes. The whole analysis was performed on BMKCloud (www.biocloud.net).RNA sequencing was accomplished by Beijing Baimaike Biotechnology Co., Ltd . Total RNA was isolated from 3T3-L1 cells using the TRIzol reagent . For RNA-sequencing analysis, three independent samples from each group, including , (group 2, LV-t-test was used to determine the p value. *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant, p  >  0.05.All the experiments were performed in triplicate and three independent repeats. Statistical analyses were performed using GraphPad Prism 8.0.2. Data are presented as mean ± SD. One-way ANOVA or two-tailed Student’s Further information on research design is available in the Peer Review FileSupplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Reporting Summary


Aspergillus fumigatus is a genetically diverse fungal species, which is near ubiquitous in its global distribution and is the major cause of the life-threatening disease invasive aspergillosis. We present 3 de novo genome assemblies that were selected to be representative of the genetic diversity of clinical and environmental A. fumigatus. Sequencing using long-read Oxford Nanopore and subsequent assembly of the genomes yielded 10–23 contigs with an N50 of 4.05 Mbp to 4.93 Mbp. Aspergillus fumigatus is a globally ubiquitous environmental mould that was recently highlighted in the World Health Organization (WHO) fungal priority pathogens list as a species of critical concern [A. fumigatus can cause invasive and chronic forms of the disease aspergillosis which results in more than 300,000 deaths per year [A. fumigatus to triazole antifungals (the first-line therapy for aspergillosis) is emerging worldwide [ concern . A. fumiper year . UnfortuA. fumigatus is genetically diverse and clusters into two clades (A and B) [A. fumigatus collection that are representative of the main diversity of A. fumigatus [34/L98H) and a single clade B isolate. Although at time of original submission, there are currently 321 A. fumigatus isolates available on NCBI [https://github.com/SJHemmings/afasont).Previous phylogenomic analysis has shown that the population of A and B) , 5. ThisA and B)  and A116A and B)  do not sumigatus  34/L98H allele, U.K.) and E142 .The isolates selected for sequencing were C6 , C87  and incubated for 48 h at 37 °C. Spores were harvested in PBS + 0.01% Tween-20 by filtration through glass wool . Spores were centrifuged (5000 rpm for 10 min) and resuspended in Yeast Cell Lysis Solution  and vortexed at maximum speed for 10 min with 1.0 mm zirconia/silica beads . The suspension was then centrifuged  and supernatant was removed and treated with RNase Cocktail™ Enzyme Mix (Thermo Fisher Scientific) according to the manufacturer’s instruction. DNA was then isolated on spin columns using AW1 and AW2 wash buffers  and eluted in nuclease free water. To achieve the required relative absorption ratios for Oxford Nanopore sequencing and to concentrate DNA, additional washing steps were carried out using 0.6X AMPure Reagent  and 70% ethanol. An SRE XS kit  was used to deplete any remaining reads below 10 kb. For quality control, DNA was visualised using Genomic DNA Screentape on a TapeStation  to ensure the average DNA length was above 20 kbp. 1 μg of DNA was prepared for sequencing using an SQK-LSK110 ligation sequencing kit  and NEBNext Companion Module  following the manufacturer’s instructions. Isolates were sequenced on a minION using an R10.4 flow cell (Oxford Nanopore Technologies) for a total of 18 h.https://github.com/SJHemmings/afasont. Finally, BLAST v2.12.0 + [Live base calling was performed using Guppy v6.3.9. Porechop v0.2.4  were use2.12.0 +  analysisAfter passing through quality control, raw reads from the minION showed coverage of 82X for C6, 73X for C87 and 149X for E142. Isolates C6, C87 and E142 were then assembled into genomes of 29,266,253 bp, 28,591,451 bp and 28,644,426 bp in length.C6 was assembled into 10 contigs with an N50 of 3.99 Mbp with a longest contig of 4.93 Mbp. AUGUSTUS  predicteC87 assembled into 23 contigs with an N50 of 2.55 Mbp, the longest contig reached 4.05 Mbp in length. AUGUSTUS  found 88E142 was assembled into 15 contigs and has a N50 of 2.67 Mbp, the longest contig was 4.20 Mbp. 8,806 protein coding genes were found by AUGUSTUS  detectedRaw read files sequenced with Oxford Nanopore Technologies and de novo genome assemblies can be accessed from the European Nucleotide Archive at EMBL-EBI under the accession code PRJEB59410. The sequence accessions for the individual assemblies are: CASBLW01 (GCA_949125545) (C6); CASBLU01 (GCA_949125165) (E142); CASBLV01 (GCA_949125185) (C87).


Swe/PSEN1ΔE9 PEL24-199 treated transgenic model of amyloid pathology to achieve this objective. PEL24-199 curative treatment induced the recovery of spatial memory and decreased the amyloid plaque load in association with decreased astrogliosis and neuroinflammation. The present results underline the synthesis and selection of a promising polyaminobiaryl-based drug that modulates both Tau and, in this case, APP pathology in vivo via a neuroinflammatory-dependent process.The progress in Alzheimer’s disease (AD) treatment suggests a combined therapeutic approach targeting the two lesional processes of AD, which include amyloid plaques made of toxic Aβ species and neurofibrillary tangles formed of aggregates of abnormally modified Tau proteins. A pharmacophoric design, novel drug synthesis, and structure-activity relationship enabled the selection of a polyamino biaryl PEL24-199 compound. The pharmacologic activity consists of a non-competitive β-secretase (BACE1) modulatory activity in cells. Curative treatment of the Thy-Tau22 model of Tau pathology restores short-term spatial memory, decreases neurofibrillary degeneration, and alleviates astrogliosis and neuroinflammatory reactions. Modulatory effects of PEL24-199 towards APP catalytic byproducts are described in vitro, but whether PEL24-199 can alleviate the Aβ plaque load and associated inflammatory counterparts in vivo remains to be elucidated. We investigated short- and long-term spatial memory, Aβ plaque load, and inflammatory processes in APP Alzheimer’s disease (AD) is a neurodegenerative disease and the leading cause of dementia worldwide. The progressive extracellular secretion of toxic amyloid-β (Aβ) peptide species further framed in amyloid plaques and neurofibrillary tangles (NFTs) formed by the intraneuronal aggregation of abnormally modified Tau protein isoforms, together with neuroinflammatory processes are the major neuropathological hallmarks of AD ,2,3. Aβ BACE1 is a key endoprotease for generating Aβ peptides starting at position 1 or position 11 (β’-cleaving site), thus representing a rate-limiting step in the process of Aβ formation . In contx-40/42 species, including the Aβ11-40/42, suggesting a selective BACE1 inhibitory activity towards the cleavage at the first amino acid residue of Aβ sequence    = 23.02,  0.0004] B. Intere 0.3601) C,D. To b 0.3601) E. The nu 0.3601) F. In PEL 0.3601) G. Overal 0.3601) H,I, impl 0.3601) .In APP/PS1 mice, increased numbers of GFAP+ astrocytes are observed after six months, especially around amyloid plaques . In the In PEL24-199-treated APP/PS1 animals, the percentage of surface in the cortex and hippocampus covered by GFAP-positive astrocyte staining was significantly reduced by more than half when compared to untreated animals B. LikewiAs the principal Tau serine/threonine phosphatase PP2A  was incrIndeed, a four-month treatment sharply increased the expression of the catalytic subunit PP2AC in the hippocampus and the cortex of the animals B, followBACE1 expression, cell localization, and activity are controlled by several factors. Since PEL24-199 has a non-competitive β-secretase activity in a cell system, we investigated whether our drug treatment did not affect the synthesis of GGA1 protein, a key β-secretase regulating factor. Golgi-localized, gamma-ear-containing, ADP ribosylation factor-binding (GGA) proteins have been shown to interact with the cytoplasmic domain of BACE1 in a phosphorylation-state-dependent manner and to regulate the endocytic trafficking of this protease . This alIn the present study, we show that the β-secretase non-competitive inhibitor compound PEL24-199 reduces amyloid pathology in the APP/PS1 model via a process that includes astrogliosis and neuroinflammation. Our results show that a curative treatment of four months in eight-month-old APP/PS1 mice with PEL24-199 restores short- and long-term spatial memory and decreases amyloid pathology and neuroinflammation in this transgenic lesional model of amyloid deposition. This curative effect is also accompanied by an increase in PP2A catalytic subunit and an upregulation in GGA1 protein levels, as previously shown.The results of the APP/PS1 model show a highly significant increase in PP2Ac phosphatase. This confirms the data from the THY-Tau22 model and shows that PP2A appears to play a major role in the beneficial curative effects mediated by PEL24-199 . This su1-40 and Aβ1-42 levels in the blood of the animals showed that PEL24-199 decreased in a non-significant extent Aβ peptides plasmatic levels in PEL24-199-treated APP/PS1 mice , with no apparent change in larger plaques (250 µm²–500 µm²). Thioflavin-S labeling revealed that large and mature amyloid deposits were reduced after the PEL24-199 treatment, while APP metabolites expression remained unchanged. PEL24-199 also did not influence total APP levels or its phosphorylation on threonine 668 ,47. TogePS1 mice , which t1-42-induced oxidative damage and inflammation and increases Aβ peptide clearance  and tissue was sonicated . A BCA assay was used to determine the total protein concentration in the TSB lysate of the hippocampus and the cortex, and each brain lysate protein concentration was diluted to a final concentration of 1mg/mL with the addition of TSB.The mice were sacrificed by beheading, and the blood was collected from the neck in heparinized tubes. Brains were removed, and one hemisphere was post-fixed for immunohistochemistry in 4% paraformaldehyde fixative diluted in PBS (pH 7.4) for a week at 4 °C and transferred to a 20% sucrose solution overnight before being frozen. The cortex and hippocampus of the other half of the brain were dissected, disposed of in a 1.5 mL isopropylene tube, and then snape-frozen by immersion of the tube in an isopropanol solution added with dry ice. Brain tissue was stored at −80 °C until used for biochemical analyses. Cortex and hippocampus were added to 200 µL of Tris-Sucrose buffer (TSB) [Tris-HCl 10 mM, pH 7.4 with 10% sucrose  and heated for 10 min at 70 °C. Using the precast 4–12% Criterion™ XT Bis-Tris polyacrylamide eighteen-well gels , 15 µg of total proteins were loaded per well, and protein electrophoresis was achieved by applying a tension of 200 V during 60 min using the CriterionTM Electrophoresis Cell using the NuPAGE MOPS SDS running buffer (1X). Apparent molecular weight calibration was achieved using molecular weight markers . Following electrophoresis, proteins were transferred from the polyacrylamide gel onto a nitrocellulose membrane of 0.4 μM pore size  using the Criterion™ blotting system by applying a constant tension of 100 V for 40 min. For APP metabolites detection (Aβ and CTFs), 16.5% CriterionTM XT Bis-Tris polyacrylamide eighteen-well gels (Bio-Rad) were used with the Tris-Tricine running Buffer (BioRad) and electrophoresis was achieved by applying a tension of 150 V for 2 h. Proteins were transferred onto a nitrocellulose membrane of 0.2 µm pore size  using the Criterion™ blotting system by applying a tension of 100 V for 30 min.v/v) (TBS-T), and 5% (w/v) of skimmed milk (TBS-M) or 5% (w/v) of bovine serum albumin (TBS-BSA) depending on the antibody. Membranes were then incubated with primary antibodies overnight at 4 °C. Conditions of use of the primary and secondary antibodies are summarized in Protein transfer and quality were determined by a reversible Ponceau Red coloration (0.2% xylidine Ponceau Red and 3% trichloroacetic acid) before an extensive wash under deionized water. Membranes were blocked for 1 h in 25 mM Tris–HCl pH 8.0, 150 mM NaCl, 0.1% Tween-20 (v/v) solution for 3 min. Sections were then blocked with normal goat serum  diluted in PBS for 1hr before incubation with a mouse biotinylated anti-Aβ antibody (4G8) at 4 °C overnight. For GFAP immunohistochemistry, sections were washed with 0.2% Triton X-100 PBS buffer for permeabilization, then incubated with a 0.3% hydrogen peroxide solution , and then blocked with 5% normal goat serum  diluted in PBS for 1hr. Sections were then incubated with rabbit anti-GFAP antibodies at 4 °C overnight before being incubated with the secondary anti-rabbit biotinylated antibody  for 1 h. After washing in 0.2% Triton X-100 in PBS, all the sections were incubated with the ABC kit (Vector Laboratories) for 2 h and developed using DAB (Sigma) before being rinsed with physiological serum. Brain sections were mounted on glass slides  and dehydrated in sequential baths of 30%, 70%, 95%, and 100% (v/v) ethanol for 5 min each. Then the slides were immersed in toluene for 15 min and fixed with mounting medium  and glass coverslips. Images were acquired using Zeiss Axioscan.Z1 slidescan and quantification of the number of plaques, discrimination by plaques size, and area covered by GFAP staining in the hippocampus of APP/PS1 mice were performed using Image J and a custom macro. The custom macro derived from the Image J Immunohistochemistry Image Analysis Toolbox was used . After adopting the DAPI contrast, the region of interest was manually defined. Blind to the images’ identification, tests were run on a few representative Aβ-labeled and Thioflavine-stained images to define the threshold to apply that encompasses the greatest number of plaques without including non-specific signals. The same procedure was run for the GFAP astrocyte staining. These defined thresholds were applied to all images and executed automatically. The ten measured parameters were as follows: Total surface of the area in mm², Total number of plaques, Plaques total surface in mm², Number of plaques 2–150 µm², Number of plaques 150–500 µm², Number of plaques 500–1500 µm², Ratio plaques area per mm², Ratio number of plaques per mm², GFAP total surface in mm², Ratio GFAP area per mm².Antibodies used in this study are listed in For immunofluorescence studies, coronal brain sections were permeabilized with 0.2% Triton X-100 in PBS and blocked with normal goat serum  diluted in PBS for 1hr before incubation with an anti-GFAP antibody at 4 °C overnight. After washes, sections were incubated with secondary antibody AlexaFluor 568 goat anti-rabbit IgG  in 0.2% Triton X-100 in PBS for 1 h. Sections were incubated with DAPI  for 5 min and mounted on glass slides. To visualize mature amyloid plaques, sections were incubated with a 1% Thioflavine-S in water solution for 30 min and then washed with sequential 80% and 90% ethanol baths. Sections were finally treated with 0.3% Suden Black (Merck Millipore 2160) for 5 min and washed with 70% ethanol to block autofluorescence. Images were acquired using a Fluorescence Zeiss Axioscan.Z1 slidescan . Quantification of GFAP staining and amyloid plaques was performed using Image J and an IHC ImageJ custom macro. The number of plaques and the area covered by amyloid plaques were calculated in the cortex and hippocampus of the APP/PS1 mice. An average of six anteroposterior sections were selected according to the Allen mouse brain atlas in four mice on average and for each experimental condition.Total RNA was extracted from hippocampi and cortex mouse brain tissue and purified using the RNeasy Lipid Tissue Mini Kit  following the manufacturer’s instructions. Extracted RNAs were quantified by the NanoDropOne spectrophotometer (Thermo Fisher Scientific) and the absorbance ratios at 260/280 nm and 260/230 nm were measured to determine the RNA purity. A total of 500 ng of total RNA were reverse-transcribed using the High-Capacity cDNA reverse transcription kit  in a final reaction volume of 20 µL. The following conditions were applied to the thermocycler: 10 min at 25 °C, 120 min at 37 °C followed by 5 min at 85 °C. The RT–qPCR reactions were performed in a Real-Time PCR System StepOnePlus (Applied Biosystems). Each reaction was performed in duplicate in 96-well plates (Applied Biosystems) with a final reaction volume of 10 μL. All reactions contained 2 μL of cDNA and 8 µL of SybrGreen mix (5 μL of Power SYBR Green PCR Master Mix (Applied Biosystems), 0.1 μL of each primer, and 2.8 μL of DEPC-treated water). The reaction protocol starts with a 10 min initial denaturation step at 95 °C, followed by forty cycles of 95 °C for 15 s, then a step at 60 °C for 25 s. Subsequently, the melting curve was verified by amplification of a single product, which was generated starting at 95 °C for 15 s, then 60 °C for 1 min and increasing by 1 °C every minute to reach 95 °C during 15 s. Each experiment included negative template controls and internal controls. Cyclophilin A was used as a reference gene, and the relative expression of target transcripts was determined by the ∆∆CT method. The sequences of primers used are given in 1-40 and Aβ1-42 were measured using ELISA kits  following the manufacturer’s instructions. Briefly, 25 µL of the samples were diluted in 175 µL of the dilution buffer. Then, 20 µL of biotin solution per well was incubated with 80 µL of the diluted samples, calibrators, and controls for 3 h at room temperature. The ELISA plate was washed using the washing buffer, and 100 µL per well of enzyme conjugate was added for 30 min. The wells were washed again, 100 µL per well of chromogen/substrate was added and the plate was incubated for 30 min in the dark. 100 µL of stop solution per well was added to stop the reaction, and the absorbance at 450 nm was measured with a Multiskan Ascent spectrophotometer . The amounts of Aβ1-40 and Aβ1-42 in the plasma were estimated by reference to the manufacturer’s standard curve and expressed in pg/mL of plasma. A minimum of seven mice per group were used for this experiment.Blood samples in heparinized tubes were centrifuged at 10,000 rpm for 15 min , and plasma was collected. Plasma levels of Aβt-test or One-Way ANOVA using Graphpad Prism Software v8 . Multiple comparisons between data series were made using Two-Way ANOVA. p values < 0.05 were considered significant.Image acquisitions and quantifications, as well as behavioral evaluations, were performed by investigators blind to the experimental condition. Results are expressed as the means ± SEM. Differences between mean values were determined using the Student’s Our results show that a curative treatment of APP/PS1 mice with PEL24-199 restores short- and long-term memory and alleviates the amyloid pathology in this mouse model, as summarized in


The immune response to pathogens is one of the organic functions at risk due to climatic change. The effects of a short-term increase in temperature on immune cell functions have been evaluated using a simple animal model, such as the sea urchin, exposed to temperatures above its comfort zone (17 °C), causing moderate and severe heat stress. After 3 and 7 days of exposure to the three temperatures tested, the coelomic fluid of the animals was collected. The coelomocytes were isolated and typed and the mitochondrial activity, the amount of lipid peroxidation, and the production of hydrogen peroxide were evaluated. These cells were also stimulated with phorbol 12-myristate 13-acetate (PMA) to evoke respiratory burst, a marker of immune response. Heat stress altered both the distribution of the various types of coelomocytes and their mitochondrial activity; it also increased lipid peroxidation and reduced the production of hydrogen peroxide. The respiratory burst occurred in the coelomocytes of all three experimental groups; however, it was greater in the group kept at 17 °C. In conclusion, thermal increase alters immune cell functions with possible repercussions on defense mechanisms against pathogens.Paracentrotus lividus adult females were randomly divided into three groups and housed in tanks at 17 °C. In two of these tanks, the temperatures were gradually increased up to 23 and 28 °C. Celomatic fluid was collected after 3 and 7 days. The coelomocytes were morphologically typed and evaluated for their mitochondrial membrane potential (MMP), lipoperoxidation extent (LPO), and hydrogen peroxide content (H2O2). Respiratory burst was induced by treatment with phorbol 12-myristate 13-acetate (PMA). HS caused a significant change in the coelomocytes’ type distribution. MMP increased in the 23 °C-group and decreased in the 28 °C-group at both 3 and 7 days. LPO only increased in the 28 °C-group at 7 days. H2O2 progressively decreased together with the temperature increase. Respiratory burst was detected in all groups, but it was higher in the 17 °C group. In conclusion, the increase in temperature above the comfort zone for this animal species affects their immune cells with possible impairment of their functions. Due to global warming, animals are experiencing heat stress (HS), affecting many organic functions and species’ survival. In this line, some characteristics of immune cells in sea urchins subjected to short-term HS were evaluated.  Global warming is a serious threat to the survival of many plant and animal species on our planet . In animMytilus galloprovincialis, disclosing morphological and functional sperm alterations associated with HS. In the sea urchin Arbacia punctulata, short-term (7 days) HS decreased gamete production and increased reactive oxygen and nitrogen species  content in the gonadal tissue .,26.25,26JC1   is a vital dye used to assess mitochondrial membrane potential. This molecule undergoes a reversible change in fluorescence emission shifting from ~595 nm wavelength (red peak), labeled as high mitochondrial membrane potential due to the formation of J-aggregates, to ~535 nm wavelength (green peak), labeled as low mitochondrial membrane potential due to the monomeric form of JC1 . Aliquot581/591 undecanoic acid -4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid)  is a fluorescent lipid peroxidation (LPO) sensor whose oxidation of the polyunsaturated butadienyl portion shifts the fluorescence emission peak from ~595 nm to ~520 nm wavelength [C11-BODIPYvelength . A 1 mM 2O2) was assessed by using the 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA)  [2O2 to the fluorescent compound DCF, whose fluorescence is proportional to the intracellular H2O2 content. A 1 mM H2DCFDA stock solution was prepared in DMSO and stored at −20 °C. Approximately 1 µL of this solution was added to the 200 µL cell sample in AFSW within the 96-well plate. After 30 min incubation at 18 °C, the supernatant in each well was removed by a multi-channel pipette and fresh AFSW was added. The samples were read with a microtiter plate reader (see above). The intracellular H2O2 levels were evaluated in arbitrary units (a.u.) on fluorescence emission peak intensity at ~525 nm wavelengths, setting the excitation wavelength at 488 nm. The positive controls were prepared by incubating the control coelomocytes with 25 μM hydrogen peroxide.The intracellular content of hydrogen peroxide  . This is−1) and stored at -80 °C. Respiratory burst was measured by fluorescence spectroscopy with a microtiter plate reader (see above) in either JC-1- or H2DCFDA-loaded cells. A standard number of coelomocytes was seeded in a 96-well plate (2 × 105/well), loaded with either JC-1 or H2DCFDA  activator phorbol 12-myristate 13-acetate (PMA) (Sigma P1585). PMA stock solution was prepared in DMSO  test. The threshold of p < 0.05 was used as the minimum level of statistical significance. Data are presented as the mean ± standard deviation (SD).Statistical analyses were performed by ANOVA by using Systat 11.0 . Before the analyses, the percentage values were transformed in arcsine, whereas for pHSmall variations  in the set and ambient (control) temperatures were recorded during the experiment by taking measurements twice a day. However, to facilitate the description, the results will referred to the set temperatures. CF did not differ among the animals exposed to different temperature conditions as reported in The pHp < 0.05) decreased compared to those harvested from the control (90.2 ± 2.1%) and 23 °C-exposed (90.4 ± 2.1%) sea urchins (p < 0.05) temperature-depended trend increasing from 1.1 ± 0.2% (17 °C) to 2.7 ± 0.2% (23 °C) and 2.2 ± 0.7% (28 °C) after 3 days (p < 0.05) inverse temperature-depended trend decreasing from 7.2 ± 1.1% (17 °C) to 5.0 ± 0.6% (23 °C) and 4.1 ± 1.4% (28 °C) after 3 days (p < 0.05) differentiated both the red and white amoebocyte content from the group exposed to 23 °C only after 7 days of treatment  increase in the MMP value was recorded in the group at 23 °C at 3 days following HS induction (24.0 ± 9.7 vs. 14.7 ± 5.6) while a significant (p < 0.05) decrease was detected in the group exposed to 28 °C for both 3 (6.4 ± 2.9 vs. 14.7 ± 5.6) and 7 (11.3 ± 3.2 vs 16.6 ± 7.9) days (p < 0.05) lowering of the MMP value was also observed in the positive control group treated with CCCP.The mitochondrial activity of the coelomocytes, evaluated as mitochondrial membrane potential (MMP), was significantly affected by HS. In fact, a significant (.9) days A. A signp < 0.05) increased only after 7 days of exposure to 28 °C (64.7 ± 5.8 vs. 54.7 ± 7.9) (p < 0.05) increase in the LPO values was also observed in the positive control treated with FeSO4 and ascorbic acid.The LPO in the coelomocytes, evaluated by fluorescent dye C11-Bodipy, significantly (7 ± 7.9) B. A signp < 0.05) decrease in H2O2 content was found in the coelomocytes at both 23 °C (192 ± 57 and 285 ± 21 a.u.) and 28 °C (172 ± 57 and 269 ± 29 a.u.) vs. 17 °C (255 ± 40 and 332 ± 26 a.u.) after 3 and 7 days from the start of the experiment, respectively  and due to the accumulation of acidic metabolites [CF are controversial. In P. lividus, a significant relationship between pHCF and seawater pH was reported but not associated with temperature variations [Arbacia punculata [CF. Our results confirm those already found in P. lividus, and demonstrate differences between P. lividus and A. punctulate; the former, in fact, together with lower thermotolerance, shows upper pHCF values of about 0.6–0.7.The rapid and progressive increase in sea temperatures related to climate change is a serious challenge to the health of marine ecosystems . Temperamination ; it mighmination ,33,34. Wr fluids . Insteadabolites . The effriations . Conversunculata , either Sea urchin coelomocytes engage in various activities ranging from immune to metabolic functions with the transport of nutrients and ensuring gaseous exchanges . These aExaiptasia diaphana and Nematostella vectensis, overnight exposure to 30 °C (moderate HS) increased phagocytic activity and immune gene expression together with cellular ROS production compared to the ambient temperature (18 °C) [M. galloprovincialis spermatozoa [Perna canaliculus in which exposure at 28.5 °C for 20 min, far above the thermal comfort zone at 7.3 °C, produced a significant increase in ROS production in hemocytes [Phagocytosis, encapsulation, and the production of ROS and RNS are the most widespread mechanisms by which the immune cells of invertebrates react under conditions of defense against pathogens and stressors . In the  (18 °C) . In the  (18 °C) ,45. MMP, (18 °C)  but also (18 °C)  as well  (18 °C) . The resrmatozoa , in whicrmatozoa . In the emocytes . HoweverPerna viridis, malondialdehyde content and MMP increased significantly under both cold and heat stress [LPO is a marker of chronic oxidative stress . It is ct stress . In our t stress  in which2O2 content takes on a different meaning in immune cells from that detectable in other somatic cells for which it represents a marker of acute oxidative stress [2O2 as one of the defense mechanisms against pathogens [2O2 content in the coelomocytes as the HS intensity increases both at 3 and at 7 days of HS could be associated with a lowering of the immune defenses of these cells. This hypothesis is further supported by the results obtained from the respiratory burst test. Respiratory burst, induced in the present study by PMA, is a strategy by which immune cells produce large quantities of H2O2 to contrast pathogens. Respiratory burst does not appear to be associated with mitochondrial activity, as found in human leukocytes [2O2. Although the coelomocytes of sea urchins under HS still responded to PMA treatment, the lower hydrogen peroxide produced compared to that obtained in the coelomocytes of animals kept under thermo-comfort conditions suggests lower immune cell function associated with heat stress.The increase in the He stress . Coelomoathogens . Therefoukocytes , althougukocytes . In the P. lividus, which holds the structure and the control of macroalgae assemblages and thus the shape of the benthic Mediterranean seascape [P. lividus sea urchins. Immune cell dysfunctions were significantly detectable already after 3 days with certain biomarkers, such as cell type distribution, mitochondrial activity, and hydrogen peroxide production, even though they became evident at 7 days of heat stress. At this time, all the above biomarkers together with coelomocyte lipid peroxidation showed marked differences between the heat stressed and control animals. These findings, together with the lower response to the experimentally induced respiratory burst, suggest that, in the females of sea urchins, the immune response is early compromised in the course of heat stress and aggravates the implications of climate change on the fitness of marine species.This study provides a new insight into the effects of the increasing temperatures on a species already heavily compromised by global warming, the sea urchin seascape . In deta