Klucnika et al. find that the DNA helicase REC repairs mitochondrial DNA (mtDNA) by homologous recombination and prevents age-associated mtDNA mutations. This study provides the first insight into the molecular machinery of mtDNA recombination in animals and demonstrates its importance in safeguarding the mitochondrial genome during ageing. Drosophila. We show that REC repairs mtDNA DSBs by homologous recombination in somatic and germline tissues. Moreover, REC prevents age-associated mtDNA mutations. We further show that MCM8, the human ortholog of REC, also localizes to mitochondria and limits the accumulation of mtDNA mutations. This study provides mechanistic insight into animal mtDNA recombination and demonstrates its importance in safeguarding mtDNA during ageing and evolution.Mechanisms that safeguard mitochondrial DNA (mtDNA) limit the accumulation of mutations linked to mitochondrial and age-related diseases. Yet, pathways that repair double-strand breaks (DSBs) in animal mitochondria are poorly understood. By performing a candidate screen for mtDNA repair proteins, we identify that REC—an MCM helicase that drives meiotic recombination in the nucleus—also localizes to mitochondria in   Preserving the integrity of the mitochondrial genome during development and ageing is crucial because the accumulation of mitochondrial DNA (mtDNA) mutations is linked to mitochondrial diseases , cancersArabidopsis thaliana mitochondria also requires RecA-related recombinases  helicase that drives the formation of most meiotic crossovers in ynthesis . Reflectynthesis . Throughynthesis . The nucynthesis . To furtynthesis . This inRECΔMTS-Halo) greatly reduced mitochondrial REC without affecting the amount of REC in the oocyte nucleus  does not impair female fertility  did not impact rescue in the soma  lethal mutant genome that carries a point mutation in mt:CoI. Flies homoplasmic for mt:ATP6[1] are healthier and more robust than ts flies, but polymorphisms in the non-coding region of the ts genome grant it a selfish transmission advantage so that it displaced mt:ATP6[1] over a few generations even at the restrictive temperature (29°C). The entire population died after several generations due to the loss of complementing mt:CoI activity from mt:ATP6[1]. However, spontaneous recombination in some lineages generated mitochondrial genomes with the functional mt:CoI from mt:ATP6[1] and the selfish drive from the ts genome, which allows these lineages to survive and propagate at 29°C  in young flies was slightly higher for controls . The incKO flies . There wKO flies  or exprent flies . We thusDrosophila lack an ortholog of MCM9 , nubbin-GAL4 (BDSC:42699), nos-GAL4 (BDSC:64308), Ubi-mtSSB-GFP , Opa1-3HA -Halo, SPN-B-Halo, SPN-D-Halo, MRE11-Halo, NBS-Halo, UASp-REC-GFP, UASp-mito-NciI, and mt:NciIresistant were generated for this paper.All fly stocks were raised on standard media at 25°C unless otherwise stated. Lines used in this study include Opa1-3HA , mt:ND2dCoIT300I , D. melaDrosophila S2R+ cells were maintained in Schneider’s medium  with 10% fetal bovine serum  and 1% penicillin-streptomycin-glutamine  at 25°C.KO cells were maintained in complete DMEM media  supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin  at 37°C. To test growth under different carbon sources, cells were maintained in DMEM supplemented with 10% FBS, 1% penicillin-streptomycin, GlutaMAX , and either 25 mM D-glucose  or 10 mM D-galactose .HeLa, Hela Cas9, HEK293T, and MCMhttps://flycrispr.org/). gRNAs were designed using FlyCRISPR target finder (http://tools.flycrispr.molbio.wisc.edu/targetFinder) and cloned into a pCFD5 plasmid. Donor plasmids were generated using InFusion Cloning . Plasmids were injected into nos-Cas9 or nos-Cas9;;REC-Halo embryos to establish individual stocks. The nature of mutations and tagging was verified by Sanger sequencing. To make transgenic lines with UASp for GAL4-driven expression, open reading frames of candidate genes were cloned into the pPWG or pPW vector. Plasmids were injected into y[1] w*;attP40 or w1118 flies to establish individual stocks. All primers used for this section are listed in Mutant generation and endogenous tagging were performed by CRISPR-Cas9-based editing as described in FlyCRISPR  containing 1% EDTA-free protease inhibitor cocktail . The homogenate was centrifuged at 17,000 × g for 10 min at 4°C to separate soluble proteins from debris. Soluble protein concentrations were determined using the Pierce BCA protein assay kit .Dissected ovaries were resuspended in PBS in parallel preparations. The harvested tissues were centrifuged at 17,000 × g for 5 min at 4°C, and the resulting supernatant was collected as the whole-cell lysate. To obtain the mitochondria fraction, the whole-cell lysate was centrifuged at 1,000 × g for 5 min at 4°C twice, and the resulting supernatant was centrifuged at 10,000 × g for 5 min at 4°C. The pellet was washed with mitochondrial suspension buffer . The final mitochondrial pellet was resuspended in a mitochondrial suspension buffer. To separate cytosolic and nuclear fractions, the whole-cell lysate was centrifuged at 1,000 × g for 5 min at 4°C. The pellet was washed twice and solubilized with mitochondrial suspension buffer + 1% NP-40 to yield the nuclear fraction. The supernatant was centrifuged at 1,000 × g for 5 min at 4°C, then at 10,000 × g for 5 min at 4°C, and the resulting supernatant was supplemented with 1% NP-40 to yield the cytosolic fraction. Soluble protein concentrations were determined using the Pierce BCA protein assay kit.To prepare whole extracts and subcellular fractions from embryos, overnight embryos were collected, dechorionated using 50% bleach and homogenized in mitochondrial isolation buffer . Homogenate was centrifuged at 100 × g for 10 min at 4°C to separate soluble proteins from cell debris. Subcellular fractions were prepared from cells using the cell fractionation kit  in the presence of 1% EDTA-free protease inhibitor cocktail. Soluble protein concentrations were determined using the Pierce BCA protein assay kit.Trypsinized cells were washed once with 4°C PBS and incubated with Pierce IP lysis buffer containing 1% EDTA-free protease inhibitor cocktail for 30 min at 4°C. Whole-cell lysates were centrifuged at 17,000 × Whole-cell lysates, subcellular fractions, and immunoprecipitates were supplemented with 100 mM Bolt sample reducing agent , incubated for 5 min at 95°C, and separated on a Bolt 4–12%, Bis-Tris gel  in MES SDS running buffer . They were transferred to Immobilon-P PVDF membranes  in a Tris/glycine transfer buffer . The blots were blocked with 10% milk for 1 h at room temperature and probed for FLAG , Halo , MCM8 , mtSSB  or TFAM . Antibodies against COX4 , tubulin , GAPDH , and histone H3  or PCNA  were used to serve as mitochondrial, cytosolic, and nuclear markers, respectively. Western blots were visualized by film exposure using horseradish peroxidase-conjugated secondary antibodies against chicken , mouse , rabbit , or rat , in combination with Clarity Western ECL substrate  on an SRX-101A developer.g at 4°C for 10 min to remove cell debris. 2–4 mg of cell extracts were incubated overnight at 4°C with 2 μg of mouse IgG , TFAM , or MCM8  antibody. Immunoprecipitates were collected with high-affinity protein G agarose beads  for 3 h at 4°C, centrifuged at 200 g at 4°C for 5 min, and washed five times with IP lysis buffer. Samples were eluted with Bolt LDS sample buffer for 5 min at 95°C, and separated on a Bolt 4–12% gel as described above.Trypsinized human cells were washed three times with PBS, and homogenized with a Dounce homogenizer together with IP lysis buffer . The lysates were incubated on ice for 20 min and centrifuged at 12,000 × Drosophila ovarian tissues were homogenized in homogenization buffer  plus 1% protease inhibitor cocktail . The cell debris and nucleus were removed by centrifuging at 500 × g for 5 min at 4°C and then 1,000 × g for 5 min at 4°C. The mitochondria were pelleted by centrifuging at 5,000 × g for 10 min at 4°C and washed at 5,000 × g for 10 min at 4°C in homogenization buffers without protease inhibitors. The mitochondrial pellet was then suspended in homogenization buffers and the protein concentration was measured using Qubit protein assay kit . For the proteinase K protection assay, 50 mg of mitochondrial proteins were pelleted by centrifugation at 5,000 × g for 10 min at 4°C and re-suspended in 500 μl of homogenization buffer, mitoplast/swelling buffer , or solubilizing buffer (homogenization buffer with 1% Triton X-100), and incubated on ice for 15 min. The mitoplast/swelling sample was pipetted up and down 15 times to disrupt the mitochondrial outer membrane. Proteinase K  was then added to the samples to a final concentration of 4 U/ml, and samples were incubated on ice for 20 min. To terminate the reaction, PMSF was added to all samples to a final concentration of 2 mM followed by 5 min incubation on ice. The resulting proteins were then precipitated by 12.5% TCA, washed with cold acetone, re-suspended in 100 μl of 1× LDS sample buffer, and boiled for 5 min. Finally, 20 μl of samples were analyzed by Western blot. Tom 20 , TIM50 , PHB2 , TFAM , and PolG1  were probed as markers for different compartments of human mitochondria. Porin , Opa1-HA , PolG1-Halo , and ATP5A  were probed as markers for different compartments of Drosophila mitochondria.The proteinase protection assay was performed as previously described . Briefly18 primers. The relative mRNA level of individual genes was measured by qPCR using 2× SensiFast SYBR Green PCR Master Mix  and normalized to ef1a. All the primers used are listed in Total RNA from whole flies, dissected tissues , or human cells was extracted using TRIzol . Extracted RNA was further purified using the Qiagen RNeasy kit with on-column DNase treatment . Purified RNA was then reverse-transcribed using RevertAid First-strand cDNA synthesis kit  and Oligo (dT)Dissected ovaries were fixed in 4% paraformaldehyde (pH 7.4) for 30 min, washed three times in PBS with 0.1% TritonX-100 (0.1% PBST), and incubated in 0.5% PBST overnight at 4°C. They were then washed once with PBS followed by Halo staining in 0.5 μM HaloTag TMR ligand  for 1 h at room temperature. To minimize background signal, ovaries were further washed three times in 0.1% PBST and incubated overnight at 4°C. If subsequent immunostaining to visualize mitochondria was required, ovaries were incubated with ATP5A antibodies  in 0.1% PBST supplemented with 3% BSA overnight at 4°C, followed by another overnight incubation with Alexa Fluor 647  in 0.1% PBST with 3% BSA. Ovaries were then mounted in Vectashield medium with DAPI  and imaged on a Leica SP8 DM6000 CS upright confocal microscope using a 63× oil lens 1.4NA and Leica LASX Acquisition software at room temperature. Images were processed using ImageJ and Affinity Designer software.5 cells were seeded into each well of a 24-well glass-bottomed plate . The next day, cells were incubated with 0.1 μM MitoTracker Red FM for 30 min, washed three times with PBS, and fixed by 4% paraformaldehyde (pH 7.4) for 10 min at 37°C. The cells were washed three times with PBS and then permeabilized by incubation with 0.1% PBST for 15 min at room temperature. Following another three washes with PBS, cells were incubated with 2% BSA in PBS for 4 h at room temperature. Primary antibodies were then added in PBS with 0.1% BSA and incubation continued overnight at 4°C. Primary antibodies used include dsDNA  and MCM8 . Following incubation with primary antibodies, the cells were washed three times with PBS and incubated with secondary Alexa Fluor 546  or Alexa Fluor 488  antibodies in PBS with 0.1% BSA for 45 min at room temperature. The cells were washed three times with 0.1% PBST and then incubated with 2 μM Hoechst and imaged on an SP5 Leica inverted confocal microscope using a 63× oil lens 1.4 NA and Leica LASX Acquisition software at room temperature. Images were processed using ImageJ and Affinity Designer software.For mammalian cells, 102O2 , 50 μM cisplatin , or PBS alone, followed by fixation and Halo staining as described above. All drugs were dissolved in PBS. Mitochondrial areas were defined by mtSSB-GFP fluorescence, and the average Halo signals in the defined mitochondrial areas were quantified using ImageJ. Egg chamber mitochondria were selected as regions of interest by thresholding of the mtSSB-GFP, and then the mean pixel intensity (AU) of the Halo channel was measured and normalized to the controls.For experiments with drug treatments prior to fixation, ovaries were dissected from flies expressing mtSSB-GFP and incubated for 2 h in PBS with 50 μg/ml bleomycin , 1 mM paraquat , 10 mM HTo measure mitochondrial membrane potential, 50,000 cells were seeded in a well of 18-well Ibidi slide  and incubated with 7.5 mM JC-10  for 30 min in media before imaging with an SP5 Leica inverted confocal microscope at Ex/Em = 490/525 nm and 540/590 nm, using a 63× oil lens 1.4NA and Leica LASX Acquisition software at room temperature, followed by ratio analysis using ImageJ.UAS-mito-NciI;REC-Halo, UAS-mito-NciI;RECΔMTS-Halo, and UAS-mito-NciI; RECKO-Halo fly lines were generated by genetic crosses to carry the following mtDNA genotypes: mt:mau/mt:mel heteroplasmy, mt:yak/mt:mel heteroplasmy, mt:ATP[1]/mt:ND2Δ1+CoIT300I heteroplasmy, or mt:NciIresistant homoplasmy. REC*-Halo is used to represent REC-Halo, RECΔMTS-Halo, or RECKO-Halo flies below. For the germline assay, heteroplasmic UAS-mito-NciI;REC*-Halo females were crossed to nos-GAL4;REC*-Halo males  and mitochondrial background . For the fertility assay, newly eclosed females of the same genotype were placed in a vial with w1118 or nos-GAL4 males. w1118 or nos-GAL4 males were used instead of REC*-Halo flies to minimize the impact of male fertility on female egg laying and hatching. The flies were flipped into a new vial each day or every 2–3 d, and the number of eggs laid and adult progeny produced in each vial were counted for a certain number of days and normalized. For the climbing assay, eight adult males of a specified age were placed in a sealed measuring cylinder, which was tapped several times to knock all the flies to the bottom. The distance climbed upwards by each fly in 30 s was recorded and the average distance climbed was calculated. For the longevity assay, 20 flies were placed in each vial on the day of their eclosion. The vials were flipped every 2 d, and survival was recorded.Prior to phenotypic assays, males of g for 10 min at 4°C. 10 μl of the sample was used for each 100 μl reaction as instructed in the kit manual. ATP levels for each sample were normalized to protein concentration measured using the Pierce BCA protein assay kit.ATP levels were measured using the ATP determination kit . Five adult males of a specified age were homogenized in 100 μl of 6 M guanidine HCl  and 0.01 M Tris-HCl (pH 7.3) and frozen in liquid nitrogen for 5 min. The sample was then incubated at 95°C for 5 min and centrifuged at 12,000 × The MCM8 mutant cell line was generated using a HeLa Cas9 line. gRNAs were designed using the IDT design tool for Alt-R CRISPR-Cas9 guide RNA. crRNA and tracrRNA were synthesized by IDT and mixed in a 1:1 crRNA:tracrRNA 30 μM duplex solution, incubated at 95°C for 5 min, cooled and diluted to 1 μM. Lipofectamine RNAiMax  was used to transfect duplexed gRNA into HeLa Cas9 cells. Single-cell clones were isolated, expanded, and genotyped by Sanger sequencing. Primers and crRNA (gRNA) sequences are listed in REC-Halo, RECΔMTS-Halo, and RECKO-Halo fly lines in an isogenic nuclear (except for the rec locus) and mtDNA background were maintained in parallel before sequencing. Total DNA of individual flies was extracted as described in g for 10 min at room temperature. DNA was recovered from the supernatant by adding 0.5× volume of isopropanol, followed by washing with 70% ethanol. DNA was then dissolved in 10 µl H2O before further dilution. Total DNA of HeLa cell lines was extracted using DNeasy Blood & Tissue Kit . DNA was prepared for sequencing using the Nextera XT DNA Library preparation kit  and index kit  following manufacturer’s instructions. Paired ends were sequenced using Illumina NovaSeq6000. Reads were aligned against the Drosophila genome (BDGP Release 6 + ISO1 MT/dm6) or human genome (hg38). Integrated Genome Viewer was used to identify mtDNA sites with variants that were above 1% read threshold for fly samples. The threshold was determined to ensure the independence of the average read coverage and number of variants. For HeLa cells, we used 3% read threshold because variants below 3% show extreme/strong strand biases, indicating a potential high false-positive rate for these SNPs. Due to the variation in genome coverage observed, mtDNA coding regions with consistently high coverage were used for analysis: 1,300–14,730 bp for fly samples; and 594–3,511, 3,629-13,631, and 13,870–16,560 bp for HeLa cells.t test or one-way ANOVA test was used to analyze the effect of a single variable, and the two-way test was used to analyze the effect of more than one variable. Dunnett’s multiple comparisons test was used after ANOVA tests to report significance. For t tests, data distribution was assumed to be normal, but this was not formally tested. Significance was defined by *P < 0.05, **P < 0.01, and ***P < 0.005.Statistical analyses were performed using GraphPad 9 software. The Student’s Drosophila ovaries .a list of candidate genes examined for the mitochondrial enrichment of their protein products when overexpressed in Click here for additional data file.SourceData F1is the source file for Fig. 1.Click here for additional data file.SourceData F3is the source file for Fig. 3.Click here for additional data file.SourceData F5is the source file for Fig. 5.Click here for additional data file.SourceData FS1is the source file for Fig. S1.Click here for additional data file.SourceData FS4is the source file for Fig. S4.Click here for additional data file.


D of 17.7 nM and 23.6 nM and low binding affinity to mouse RBC with an estimated KD of 335 nM and 528 nM for RB12 and RE8, respectively. Two derivative sdAbs, RMA1, and RMC1, with higher affinities against mouse RBC, were generated via affinity maturation . Pharmacokinetic investigations in mice demonstrated prolonged circulation half-life of an anti-RBC-TNF-α bispecific construct (75 h) compared to a non-RBC binding control (1.3 h). In summary, the developed anti-RBC sdAbs and fusion constructs have demonstrated high affinity in vitro, and sufficient half-life extension in vivo.Small therapeutic proteins are receiving increased interest as therapeutic drugs; however, their clinical success has been limited due to their rapid elimination. Here, we report a half-life extension strategy via strategy via red blood cell red blood cell (RBC) hitch-hiking. This manuscript details the development and characterization of novel anti-RBC single-domain antibodies (sdAbs), their genetic fusion to therapeutic antibody fragments (TAF) as bispecific fusion constructs, and their influence on TAF pharmacokinetics and biodistribution. Several sdAbs specific to the band 3 antigen were generated via phage-display technology. Binding affinity to RBCs was assessed via flow cytometry. Affinity maturation via random mutagenesis was carried out to improve the binding affinity of the sdAbs. Bi-specific constructs were generated by fusing the anti-RBC sdAbs with anti-tissue necrosis factor alpha (TNF-α) TAF via the use of a glycine-serine flexible linker, and assessments for binding were performed via enzyme-linked immunosorbent assay and flow cytometry. Pharmacokinetics of anti-RBC sdAbs and fusion constructs were evaluated following intravenous bolus dosing in mice at a 1 mg/kg dose. Two RBC-binding sdAbs, RB12 and RE8, were developed. These two clones showed high binding affinity to human RBC with an estimated K Biotherapeutics have gained outstanding success in recent years, with more than 200 approved for clinical use and more than 1000 currently in clinical development , the smaBand 3 protein (b3p), also known as anion transport protein 1 (SLC4A1), is expressed primarily in the RBC membrane and, to a lesser extent, on the basolateral face of collecting ducts of the nephron . B3p conL , leading to good solubility and stability [The recent discovery of a unique antibody class devoid of light chains in camelids and cartilaginous fish  has ignited the interest in developing alternative immunoglobulin scaffolds ,8. Thesetability . The VHHtability ,11,12. TIn preparation for llama immunization, bio-panning, and screening, RBC b3p was extracted from human RBC. SDS-PAGE gel analysis confirmed a prominent band of the b3p at around 95 kDa with an acceptable purity A. Each 1D values of 17.7 nM and 23.6 nM for RB12 and RE8, respectively. The binding affinity to mouse RBC was lower than to human RBC, with estimated KD values of 335 nM and 528 nM 3 linker. The bispecific constructs have molecular weights of approximately 33 kDa  TAF via a flexible glycine-serine  of RBC-binding sdAbs, hematological analysis was performed in mice. As shown in RBCs have been utilized previously in drug delivery strategies due to their unique and convenient properties, including homogeneity, abundance, long circulation half-life, remarkable endurance and resilience to external modification, and lack of nuclei and intracellular organelles . There aInitial efforts aimed to target surface antigens for coupling therapeutics to RBCs targeted complement receptor type 1 (CR1), which functions as an anchorage site for immune complexes containing activated complement C3b . SeveralGlycophorin A (GPA) has also been explored as an RBC surface protein target for use in half-life extension strategies. GPA is a membrane-bound sialoglycoprotein of the human erythrocyte membrane, which contributes to RBC structural integrity and surface charge . The expB3p is the most abundant RBC membrane protein with 1.2 million copies/cell, accounting for 25% of the total membrane protein. It is expressed selectively in RBC ,29; in aIn the Camelidae family, IgG antibodies consist of three isotypes: IgG1, which has the conventional structure composed of both light chains and heavy chains, and the heavy-chain only IgG2 and IgG3 . Due to In several rounds of panning and screening, several sdAb clones were identified that bind to human b3p and human RBC, but only 2 of those clones, RE8 and RB12, also bound to mouse RBC. Since we wished to demonstrate proof-of-concept studies in mice, these two clones were selected for further characterizations and optimizations. Assessment via flow cytometry showed that both sdAbs had high affinity against human RBC but low affinity against mouse RBC. Clones with higher affinity to mouse RBC were obtained through mutagenesis and the affinity maturation process; however, enhanced binding to mouse RBC was accompanied by loss of binding to human RBC. Although it would be desirable for constructs to have high binding to both mouse and human RBC, the species selectivity of RBC binding is consistent with binding selectivity of the sdAbs to b3p. The lead sdAb clone RMC1 was selected to generate anti-RBC fusion constructs.Small therapeutic proteins have been developed for many therapeutic applications, including T-cell retargeting , neutralIn conclusion, the present work developed and evaluated anti-RBC sdAb and bispecific constructs. The anti-RBC sdAb exhibited slow clearance from the systemic circulation, which is a desirable attribute for our half-life extension strategy. Future investigations will evaluate the benefits of the anti-RBC strategy in improving the therapeutic activity of small protein constructs.g for 5 min. The washed cells were mixed with ten volumes of ice-cold 5P8 buffer  containing 0.2 mM DTT, 20 ug/mL PMSF, and incubated for 10 min on ice. The cell lysate was centrifuged for 20 min at 27,000× g at 4 °C, and hemolysate was removed by aspiration. The ghost cell pellet was washed for an additional four times with 5P8 buffer.B3p was extracted from human RBC via a previously described method . Human Rg for 20 min at 4 °C. The pellet was resuspended with a syringe in 10 vol of SE buffer, incubated at 37 °C for 30 min, then centrifuged at 46,000× g for 20 min at 4 °C. This process was repeated one more time before resuspending the spectrin-depleted ghost membrane with a syringe in 10 vol of KI extraction buffer and incubating for 30 min at 37 °C. The ghost membrane was pelleted by centrifugation for 30 min at 46,000× g at 4 °C, then resuspended in 5P8 + PMSF buffer, and incubated at 4 °C for 10 min. The ghost membrane was centrifuged at 46,000× g for 30 min and washed once more time with 5P8 + PMSF buffer. To solubilize the KI-extracted ghosts, 4 vol of 1% C12E8 (v/v) in 228 mM sodium citrate pH 8.0 + 1 mM DTT + PMSF was added, and incubated for 20 min on ice. The resulting suspension was centrifuged at 46,000× g for 30 min  at 4 °C, and the supernatant was collected and further purified with affinity chromatography following published procedures [The ghost cell membrane was washed with 10 vol of ice-cold SE buffer, then centrifuged at 46,000× ocedures . B3p isoTo establish a single domain antibody (sdAb) phage display library, a llama provided by Capralogics  was subcutaneously immunized with 300 μg of extracted human b3p in incomplete Freund’s adjuvant every three weeks for a total of four immunizations. Ten days after the fourth immunization, 600 mL of freshly harvested blood was used to build a phage-displayed library as described previously .12 c.f.u. (colony forming unit)/mL in blocking buffer (2% milk PBS), and 100 µL of the diluted phage was distributed into each well and incubated for 2 h at room temperature. For the subsequent panning, the stock phage was diluted 1:1 in the blocking buffer. After the incubation, the plate was washed with PBST (PBS + 0.05% Tween 20) 5 times, 10 times, 15 times, and 15 times for the 1st, 2nd, 3rd, and 4th rounds of panning. Bound phages were then eluted by incubation with 100 µL of 1 mg/mL trypsin for 30 min at room temperature. Output phage was titrated and re-infected with TG-1 cells for phage production in the subsequent round of panning.To enrich positive binders, 50, 25, and 10 µL of 10% human RBCs were diluted in PBS buffer for the 1st, 2nd, and 3rd rounds of panning, respectively. Cells were washed three times with PBS and blocked with MPBS (PBS + 5% non-fat dry milk) at RT for one h. Following washing with PBS, for the first panning input, the stock phage was diluted to 10w/v) glucose), and incubated overnight at 37 °C. A master plate was generated by inoculating a single colony into wells of a 96-well round-bottom culture plate filled with 100 µL of 2xYT supplemented with 100 µg/mL ampicillin, 2% (w/v) glucose, and 10% (v/v) glycerol, and grown overnight at 37 °C. These wells were then used to inoculate wells of 96-deep well plates containing 1 mL of 2xYT medium (100 µg/mL ampicillin + 0.1% (w/v) glucose per well). Plates were incubated for three hours at 37 °C and 250 rpm until OD600 ≈ 0.5. Nanobody expression was induced with 4 µL of 0.5 M IPTG per well  and incubated for an additional 15–24 h at 37 °C and 300 rpm.Phage-infected TG1 cells were grown overnight, serially diluted in LB, spread over individual culture plates containing selective medium 3 linker. The DNA of sdAbs and bispecific constructs was ligated to a cut expression plasmid PET-22b and transformed to the E. Coli Shuffle T7 cells. A single colony was inoculated, and overnight grown culture was diluted 1:100 to TB medium. The cells were grown at 30 °C until OD600 = 0.6–0.8, and 1 mM IPTG  was added to the culture to induce expression. After a 20-h expression, the protein in cell pellets was extracted by Bugbuster buffer  and purified by Ni-NTA resin  by following the manufacturer’s protocol. The sdAb was then dialyzed into loading buffer  and purified using a Bio-Scale Mini Ceramic Hydroxyapatite Multimodal Chromatography Type I (CHT) Cartridge . Briefly, the CHT resin was pre-equilibrated with 3 column volumes (CV) of elution buffer  and then equilibrated with 10 CV of loading buffer at a rate of 2.0 mL/min. The sample was then loaded onto the column at a rate of 2.0 mL/min followed by 5 CV of loading buffer. The elution process consisted of gradients at a rate of 2.0 mL/min of 20 CV of 0–100% elution buffer. Fractions were collected during the elution process based on an A280 ≥ 0.05. Antibody purity was assessed by SDS-PAGE analysis. Briefly, antibody samples were prepared in non-reducing buffer with boiling for 5 min. Samples were then loaded on an SDS-PAGE gel (Bio-rad), and electrophoresed at 120 V for 30 min. The gel was stained with Coomasie blue R-250 (Bio-Rad) and destained with double distilled water following the manufacturer protocol, and the migration distances of different proteins were compared to the migration distances of molecular weight standards.An anti-TNF-α sdAb sequence taken from Beirnaert et al.  was fuseg for 10 min, and plasma and the buffy layer were discarded. The remaining mouse RBCs were washed thrice and resuspended in PBS (500× g 10 min). The commercial human or isolated mouse RBCs were counted and washed once with PBS + 1% Bovine serum albumin (BSA), and 1 × 106 cell aliquots were dispensed into each 5 mL flow cytometry tube . Cells were then pelleted by centrifugation (300× g for 5 min), resuspended into 300 µL of induced cell culture medium, and incubated for 1 h at 4 °C. Cells were washed two times with 1 mL of washing buffer each time and then incubated with 300 µL of washing buffer containing mouse anti-His-tag secondary antibody  diluted 1:500 v/v for one hour at 4 °C. To label the antibodies, 250 µL of PE-conjugated goat anti-mouse polyclonal antibodies were diluted at 1:250 v/v for 30 min at 4 °C. Samples were washed, resuspended in 500 µL of washing PBS on ice, and protected from light until flow cytometry analysis . Purified anti-RBC sdAbs or fusion constructs at a range of concentrations from 0.03 nM to 1000 nM were analyzed to generate binding curves, and binding affinity (DK) was estimated using GraphPad Prism software  using the equation below:maxB is the binding capacity, DK is the binding affinity, and h is the Hill slope.ELISA-positive clones  were tested for binding to red blood cells via flow cytometry. Briefly, whole blood from Swiss Webster mice  was collected in ethylenediaminetetraacetic acid (EDTA) and spun at 1000× 125I) following a modified chloramine-T method described previously [125I  and 20 µL of 1 mg/mL chloramine-T for 90 s, then 40 µL of 10 mg/mL potassium iodide was added to terminate the reaction. Subsequently, 125I labeled antibodies were separated from the mixture via gel filtration . Purity and radioactive activity of the 125I-antibodies were assessed via thin layer chromatography  and gamma counting , respectively.Antibodies were radiolabeled with iodine-125  and RBCs (~25 µL/sample) were separately precipitated with trichloroacetic acid (TCA). BSA (200 µL of 1%) in PBS and TCA (700 µL of 10%) were added to samples, followed by 15-min incubation on ice. The precipitate was isolated by centrifuging at 14,000 rpm for five minutes and discarding the supernatant. The precipitate was then washed three times with PBS. Radioactivity from plasma and RBC TCA precipitation was counted using a gamma counter and corrected for background radioactivity, gamma counter efficiency, and radioactive decay. Free and RBC-bound antibody concentrations were determined from plasma radioactivity and RBC radioactivity, respectively. Whole blood antibody concentration was calculated, assuming a hematocrit of 44.6%. Non-compartmental analysis was performed in Simbiology (Matlab software).For the pharmacokinetic studies of the anti-band-3 sdAbs , doses of 1 mg/kg of unlabeled antibodies and 400 µCi/kg tracer doses of iodinated antibodies were administered via a penile vein to three homozygous Nu/J mice  or Swiss Webster mice. Blood was collected from the retro-orbital sinus, and blood samples (50 µL/sample) were centrifuged at 200× n = 5). Blood was sampled through retro-orbital collection prior to dosing for the determination of baseline measurements of hematological parameters. Additional samples were obtained at several time-points up to 28 days post-dosing. Blood samples were collected using EDTA-coated capillary pipette tubes. Red blood cell concentration, hemoglobin concentration, and hematocrit were determined using a VetScan HM5 Hematology Analyzer , normalized by the baseline measurements, and reported as a percentage of pretreatment values. In addition to hematological parameters, body weight change was also monitored and analyzed.For the hematology study of anti-RBC sdAbs RMC1 and RB12, Swiss Webster mice were dosed at 5 mg/kg via retro-orbital injection  were then TCA-precipitated and radioactivity counted with corrections for background radioactivity, gamma counter efficiency, and radioactive decay. It is assumed that the ratio of free vs. protein-labeled radioactivity in the wash buffer is the same as that in the tissue.For the biodistribution study of RMC1-aTNF and 1HE-aTNF (served as a control), Swiss Webster mice were dosed at 1 mg/kg through IV administration with a tracer dose of


After the induced Tregs (iTregs) were confirmed to suppress lymphocyte proliferation and pro-inflammatory T help cells (Th1 and Th17) activation, a chitosan-stabilized nanoparticle drug delivery system (NDDS) was developed according to the optimized formula of IL-2/TGF-β/AS. In vivo study, the NDDS was injected into the knees of mice with collagen-induced arthritis (CIA). As a result, the NDDS remarkably reduced the pathological score of the CIA, alleviated the inflammatory cell infiltration and synovial hyperplasia, and minimized cartilage tissue damage in the knee joint of the CIA mice. Mechanically, the NDDS administration promoted Treg differentiation and decreased Th17 production, consequently reversing the ratio of Treg/Th17, and reducing the secretion of TNF-α in the sera, which facilitated to relieve the severity and progression of arthritis. In sum, NDDS capable of efficiently inducing Tregs were constructed successfully and provided a potential platform for treating RA by restoring the equilibrium of Treg/Th17 destroyed in RA.Regulatory T cells (Tregs) display great promise in rheumatoid arthritis (RA) therapy. However, their low number and differentiation rate limit their further application in the clinics. In the present study, we first optimized a combination of IL-2, TGF-β and cyclin dependent kinase inhibitor AS2863619 (IL-2/TGF-β/AS), which could induce Tregs with high efficiency  Treg-targeted nanoparticle drug delivery system rescues the imbalance of Th1/Th2 and Treg/Th17 impaired in rheumatoid arthritis. Thus, redundant Th1 and Th17 cells impair the equilibrium of Th1/T helper 2 cell (Th2) and Tregs/Th17 cells, respectively, which is usually in dynamic balance under the physiological conditions. To this end, tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), and interleukin-17A (IL-17A) are secreted in tremendous amounts, which puts the joint continuously in a state of chronic inflammation [Rheumatoid arthritis (RA) is a persistent, systemic inflammatory disease, characterized by inflammation of the synovial membrane of the joint cavity, the lining layer thickening, and significant lymphocyte infiltration, eventually resulting in joint deformity and loss of function ,2. Thougnt of RA ,4. Primeammation ,6, and cammation . In the ammation , glucocoammation . Due to ammation .+T cells and NK cells and the activation and proliferation of autoreactive T cells by producing inhibitory cytokines, such as transforming growth factor-β (TGF-β) and interleukin-10 (IL-10) [. Consequently, Tregs have been a target of cell-based therapies with new biological medicines to treat RA [Tregs are characterized by strong immunosuppressive properties to maintain immunological homeostasis by controlling peripheral immune tolerance. They are identified by the expression of Foxp3, CD4, and CD25. Through intercellular interaction, Tregs consistently block the cytotoxic effects of CD8 (IL-10) . It is n (IL-10) . Accumul (IL-10) . Consequtreat RA .To our knowledge, interleukin-2 (IL-2) and TGF-β are the significant agents employed to generate iTregs. However, the activity of iTregs following expansion is obviously impacted by the prolonged induction duration and low induction efficiency . AS28636With the development of biomaterials, drug delivery strategies for treating disease have been substantially well-designed and applied. Reasonable drug “packing” can not only enhance its pharmacokinetics as well as its solubility and bioavailability, but also reduce side effects by controlling their release speed ,17. A naIn the present study, we first set up an optimized combinations with IL-2, TGF-β and AS2863619 (IL-2/TGF-β/AS) to induce mouse Tregs at high efficiency. Then, chitosan-stabilized BSA nanoscaled particles loaded with the formula of IL-2/TGF-β/AS were applied to fabricate a nanoparticle drug delivery system (NDDS). As last, the therapeutic effect of the nanoparticles was explored by injected to the knees of the mice with collagen-induced arthritis (CIA). Upon the administration of the NDDS, the severity of arthritis was mitigated, demonstrated by the reduced the pathological score, the decreased inflammatory cell infiltration and synovial hyperplasia . Mechani22.1DBA/1 mice and BALB/c mice were obtained from SLAC LABORATORY ANIMAL . Six-week-old male mice were used for all experiments. All animal studies were approved by the Subcommittee on Research and Animal Care (SRAC) of Soochow University and were performed by following the guidelines of the Experimental Animal Center of Soochow University. The source of the reagents and antibodies used in this study were listed in the 2.2+T cells were sorted from the spleen of the DBA/1 mice with mouse CD4 MicroBeads  and cultured in the complete RPMI 1640 medium . Sorted CD4+T cells were stimulated with Ultra-LEAF™ purified anti-mouse CD3ε and CD28 antibodies . Tregs were induced by adding corresponding reagents, IL-2, TGF-β and/or different concentrations of AS for 48 h.CD42.3Cells were collected and stained with anti-CD4, CD25 and Foxp3 antibodies following the instruction of the manufactory. For intracellular IFN-γ, IL-2 and IL-17A staining, the cells were stimulated in Cell Activation Cocktail at 37 °C for 4 h before being stained with the antibodies against to the markers. Stained cells were read on flow cytometry , and data were analyzed with FlowJo V10 software .2.4−ΔΔCt method. The sequence of the primers was listed in the Total RNA was extracted by Trizol and quantified with NanoDrop . Then, RNA was reverse-transcripted into cDNA and subjected to qRT-PCR with SYBR Quantitative Premix Kit . The relative RNA expression (fold changes) of target genes were normalized by house-keeping gene GAPDH and calculated with the 22.5+T cells were purified from the spleens of DBA/1 mice, and Tregs were induced with IL-2 (10 ng/mL), TGF-β (10 ng/mL) and AS (0–1000 nM). Ficoll-isolated lymphocytes (LYM) were extracted from the splenocytes of BALB/c mice and labeled with CFSE (5 μM). Then, iTreg and LYM were co-cultured at the ratio of 0:1, 1:8, 1:4, 1:2, and 1:1, respectively. After 72 h of culture, the proliferation of lymphocytes and activated T cells were detected by FCA.CD42.6+ T cells were detected by FCA.Tregs were induced for 48 h and harvested. Some of the iTreg cells were treated with mitomycin C (20 μg/mL). The others were further cultured in the culture medium deprived of cytokines, and the supernatant of iTregs was collected after 24 h. Meanwhile, lymphocytes prepared from the spleen of DBA/1 mice were co-cultured with mitomycin C-pretreated iTreg or the supernatant of iTregs with IL-2 (20 ng/mL), IL-6 (20 ng/mL), IL-12 (20 ng/mL) and TGF-β (5 ng/mL). Cultured for 48 h, the cells were collected, and IFN-γ and IL-17A in CD42.7The nanoparticle drug delivery system was fabricated according to the previous work . In brie2.8Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used to observe the morphology of nanoparticles and NDDS. The average particle size of nanoparticles and NDDS was detected by Mastersizer .To detect the cytokine-released profiles of NDDS, the supernatant of NDDS was collected at 1, 2, 6, 12, 24, 48, 72, 96 and 120 h, respectively. The supernatant was placed in a quartz cuvette and the released rate of AS was detected at 285 nm by UV spectrophotometer. Meanwhile, the release curve of IL-2  and TGF-β  were assayed by Enzyme-Linked Immunosorbent Assay (ELISA) according to the instruction of the manufactory.To investigate the biocompatibility of the NDDS, the CFSE-labeled lymphocytes from DBA/1 mice were cultured in the cell medium, or medium soaked in nanoparticles and NDDS. The proliferation of lymphocytes was detected on days 1, 3 and 5 by FCA.+T cells from the DBA/1 mice sorted by magnetic beads were cultured in the leaching solution of the nanoparticles or NDDS, and the proportion of iTreg cells was determined by FCA 48 h later.To study the biological characters, the nanoparticles or NDDS were soaked in the complete RPMI 1640 cell medium, and 48 h later, the leaching solution was harvested. CD42.9CIA was generated as described previously . BrieflyDAB/1 mice were induced into CIA. On days 28 and 33, the CIA mice were divided into four groups randomly (n = 5 for each group) and injected with 20 μL different solutions in the joint cavity of each hind limb for intervention. The CIA-PBS group was injected with PBS, the CIA-NPs group with PBS solution containing nanoparticles, the CIA-Free group with PBS solution containing IL-2 (24.68 μg/mL), TGF-β (24.68 μg/mL) and AS (2.47 mM), and the CIA-NDDS group with PBS containing NDDS loaded IL-2 (35.25 μg/mL), TGF-β (35.25 μg/mL) and AS (3.41 mM) according to the released profiles.2.10On the 21st day after immunization, the arthritis score and body weight of DBA/1 mice were evaluated every there days. The redness and swelling of the front and rear limbs of mice were observed. Each paw was scored individually based on a 0–4 scale and the maximum score of one mouse was recorded as a score of 16 . The criOn day 38, the mice were euthanatized and tissues were collected. The joints were fixed, embedded, sliced and stained with Hematoxylin-eosin (H&E) staining . In addition, Safranin-O/Fast Green  was applied to stain cartilage tissues.2.11+T lymphocytes, Th1, Th2, Tregs and Th17 were analyzed by FCA. At the same time, the serum TNF-α level was detected by ELISA .Mononuclear lymphocytes from the lymph nodes and spleens of CIA mice were isolated and the subpopulations of CD42.12Followed by the previous study , bone hi2.13The data were denoted as mean ± standard deviation (SD). Statistical analysis was performed with GraphPad Prism 7 (USA). Statistical significance was assessed by Student's t-test (two groups) or One-way ANOVA (more than two groups). The value of P < 0.05 was considered to be statistically significant.33.1+T cells from the spleen of DBA/1 mice by magnetic beads. In order to determine the optimal combination for inducing Tregs at high efficiency, CD4+T cells were cultured with IL-2, TGF-β, and different concentrations of AS. Forty-eight hours later, FCA data revealed that the addition of IL-2 alone was unable to induce Tregs, while IL-2 mixed with TGF-β or AS alone was able to induce Tregs. The effect of AS combined with IL-2 and TGF-β outperformed the effect of AS alone  and Th17 (marked as CD4+IL-17A+) cells. However, upon the addition of the iTregs or the culture supernatants, the frequencies of Th1 and Th17 cells dropped significantly  and TGF-β . Therefoenotypes . Meanwhificantly C–F. In a3.3+T cells from the DBA/1 mice 38 days after initial immunization and treated with IL-2/TGF-β/AS. Compared with normal DBA/1 mice, the proportions of Th1, Th2, iTreg, and Th17 cells in CIA mice were higher than those in normal mice  and a chitosan-stabilized nanoparticle drug delivery system with IL-2/TGF-β/AS (NDDS) by desolvation technique following the published paper . Their mWe explored the released profiles of the cytokines by measuring the amounts of the cytokines in the supernatant at the indicative times. The release rates of AS, IL-2, and TGF-β at 96 h were 71.48 ± 0.41%, 61.77 ± 7.85%, and 61.59 ± 6.26%, respectively, as shown in Then we cultured lymphocytes in the leachate of the NPs and investigated the proliferation of cells. The data showed that there was no significant difference in lymphocytes proliferation between the different groups E. Additi+T cells isolated from the splenocytes of DBA/1 mice with NPs or NDDS leaching solutions, and the results revealed that the differentiation ratio of iTreg cells generated by the two solutions was 7.59 ± 0.71% and 63.23 ± 3.31%, respectively (Of expectation, we treated CD4ectively , Fig. S23.5The CIA was induced successfully by immunization of DBA/1 mice on days 0 and 21 . NDDS tB. NDDS rThe joint score scale is one of the indications for the progression of the disease and the function of the joints . The higWhen CIA was successfully induced in DBA/1 mice, the joints displayed redness, swelling, and immobility. Redness and swelling were faded when the intervention was performed D. The kn3.6The subchondral bone mass loss often happens in CIA . It was 3.7TNF-α has a significant function in mediating arthritic damage and may exacerbate the severity and course of RA illness . CIA mic+T cells in the spleen and lymph nodes was analyzed. As found in The joint deterioration in CIA mice may eventually result from immune system malfunction and invading effector cells. By FCA, the subtype population of CD44Generally, immune tolerance is a complex dynamic process that involves a coordinated network of immune cells and molecules. It is regulated by pro-inflammatory and anti-inflammatory factors in the microenvironment, such as Th1/Th2, Treg/Th17, which are usually in a balance in the physiological condition. If pro-inflammatory factors are predominant, the balance is disturbed and autoimmune diseases develop. Thus, creating a favorable environment for the physiologically dynamic immunological balance is a promising strategy for treating autoimmune diseases.+T cell differentiation in favor of Tregs. Our vitro results displayed that Tregs induced by IL-2/TGF-β/AS had an immunosuppressive function both in the physiological and inflammatory environment, which is consistent with relevant studies [Recently, the research on the regulation of Treg/Th17 cell balance has drawn a great amount of sight since the ratio of Treg/Th17 cells shifting to Tregs is favorable to treat autoimmune diseases ,37. Thus studies . Thus, tin vivo directly. With the help of biomaterials, sustainable and controlled release of drugs becomes possible [Released burst and rapid diffusion are other concern when free drugs are delivered possible . Researcpossible . Fan N epossible . Studiespossible . HoweverIn vitro experiments, we found that the chitosan-stabilized BSA vector was homogeneous in size, had good biocompatibility, and had no effect on the activation and proliferation of T cells.In this study, we constructed NDDS by means of the desolvation technique following the previous research ,44, and Compared with the normal DAB/1 mice, we found that the imbalance of Th1/Th2 and Treg/Th17 existed in the mice with CIA, which contributed to the inflammatory environment of RA. Consequently, the inflammatory environment attributed to degrade chondrocytes and destroy subchondral bone microstructure, which was evidenced in our study. Notably, after NDDS injection, we found that NDDS better alleviated the lesions of CIA in mice as well as bone loss, compared with other groups. It may be because the sustained IL-2/TGF-β/AS release increased the proportion of Tregs dramatically and decreased the proportion of Th1 cells, Th17 cells and secretion of TNF-α, thereby shifting the imbalance of Th1/Th2 and Treg/Th17 to a less inflammatory phenotype. Thus, NDDS displays a promising therapeutic effect in RA.+T subtype cells. Other immune cells, such as CD8+T subpopulation cells, are also needed to be further investigated. Meanwhile, in order to enhance NDDS’ therapeutic effect and minimize the side effects of drugs, we need to make a more detailed exploration on the frequency and dose of drug administration with biomaterials.However, there are some limitations in our study. We have only explored the effect of this combination of IL-2/TGF-β/AS on CD45In the present study, we screened out the optimal combination of IL-2, TGF-β, and AS that high-effectively induced iTreg cells. We further constructed NDDS and injected them locally into the knee joint to alleviate the disease of CIA in mice. Mechanically, the NDDA administration promoted Treg differentiation, consequently restored the equilibrium of Treg/Th17, and decreased the secretion of TNF-α in the sera. This study provides a new immunotherapy strategy for RA as well as other autoimmune diseases, but the deep biological mechanism and clinical translation need to be further studied.Lin Wang: Conceptualization, Methodology, Writing- Original Draft, Data Curation, Investigation. Yi Wang: Methodology, Data Curation, Software. Chang Liu: Data Curation, Formal analysis. Jiachen He: Data Curation, Validation, Formal analysis. Xu He: Data Curation, Validation. Xiongjinfu Zhang: Data Curation. Can Zhu: Data Curation, Validation. Jie Sun: Conceptualization, Project administration, Supervision. Qin Wang: Project administration, Supervision, Resources. Hao Chen: Project administration, Supervision. Qin Shi: Methodology, Conceptualization, Resources, Writing- Review & Editing, Project administration, Supervision.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Jup−/− AC model, (Jup being the gene coding for PG) mice, elevated levels of p38MAPK, an activator of ADAM17, were found. However, ADAM17 levels were unaltered in Jup−/− mice hearts. Nonetheless, inhibition of ADAM17 led to enhanced cardiomyocyte cohesion in both Jup+/+ and Jup−/− mice, and in HL-1 cardiomyocytes. Further, enhanced cohesion in HL-1 cardiomyocytes after acute inhibition of ADAM17 was paralleled by enhanced localization of DSG2 and DP at the membrane, whereas no changes in desmosomal assembly or the desmosomal complex were observed. In conclusion, acute inhibition of ADAM17 might lead to reduced cleavage of DSG2, thereby stabilizing the desmosomal adhesion, evidenced by increased DSG2 and DP localization at cell borders and eventually cardiomyocyte cohesion. We believe that similar mechanisms exist in AC.A Disintegrin And Metalloprotease (ADAM) family proteins are involved in several cardiac diseases, and some ADAMs have been associated with cardiomyopathies. ADAM17 is known to cleave desmoglein 2 (DSG2), one of the proteins involved in the pathogenesis of arrhythmogenic cardiomyopathy (AC). Desmosomal stability is impaired in AC, an inheritable genetic disease, the underlying causes of which can be mutations in genes coding for proteins of the desmosome, such as DSG2, desmoplakin (DP), plakoglobin (PG), plakophilin 2 or desmocollin 2. Stabilizing desmosomal contacts can therefore be a treatment option. In the heart of the murine  Members of the A Disintegrin And Metalloprotease (ADAM) family are transmembrane proteins with proteolytic activity, that can be involved in cancers, cardiac diseases, diseases of the central nervous system or the liver . So far,1-adrenergic receptor, but also proteins involved in cell cohesion, i.e., DSG2 or collagen XVII (Dsg2−/− murine model for arrhythmogenic cardiomyopathy (AC), ADAM17 levels were increased in the hearts of 2 week old mice  converting enzyme (TACE) are known, including the epidermal growth factor (EGF), the transforming growth factor α (TGFα), the βgen XVII . In H9C2gen XVII . Increasgen XVII , whereasgen XVII . ADAM17 gen XVII . Furthergen XVII . Apart fgen XVII . A deficgen XVII . Finallyold mice .AC is a genetic disorder that leads to a fibro-fatty replacement of cardiac muscle tissue. Untreated, AC can lead to sudden cardiac death. Underlying causes of the disease include mutations in genes coding for proteins of the desmosome, i.e., DSG2, desmocollin 2 (DSC2), plakophilin 2 (PKP2), desmoplakin (DP) or plakoglobin (PG), but also proteins not associated with the desmosome can be affected. Apart from weakened cardiomyocyte cohesion, cardiomyocyte apoptosis and necrosis, fibro-fatty replacement of the myocardium, and arrhythmias, AC also can lead to inflammation of the cardiac tissue, which damages the affected tissue further . The infInhibition of ADAM17 might be beneficial in the setting of AC as seen in other cardiovascular diseases . Increas2 for 4 days in medium without norepinephrine.Immortalized murine cardiomyocytes (HL-1 cardiomyocytes), provided by William Claycomb , were cultivated as described before . For expJup−/− mice were obtained as described before  for 90 min. After changing the medium back to Ca2+-containing medium and incubation with the respective mediators, experiments were performed.Samples were treated for 90 min with the ADAM17-inhibitor TAPI-1  dissolved in DMSO. For CaTM Protease Inhibitor Cocktail, Roche, #CO-RO) and phosphotase  inhibitors, scraped into reaction tubes and sonicated.For Western blots, samples were washed with PBS on ice and lysed into SDS lysis buffer with protease  containing protease and phosphatase inhibitors. The lysate was then transferred to gentleMACS™ M-tubes , and dissociated using the protein_01_01 program of the gentleMACS™ OctoDissociator .For immunoprecipitations, cells were cultured in T75 flasks and after treatments lysed for 30 min on ice on a rocking platform into RIPA lysis buffer  was used to measure protein concentration according to the manufacturer’s protocol.After lysate denaturation in Laemmli buffer for 10 min at 95°C, lysates were loaded on SDS-PAGE gels together with the PageRuler™ Plus Prestained Protein Ladder . Transfer to nitrocellulose membranes  was achieved using the wet-blot method. After blocking, membranes were incubated with primary antibodies overnight at 4°C: anti-ADAM17 , anti-DES , anti-DP , anti-DSG1/2 , anti-pEGFR845 , anti-pEGFR1068 , anti-EGFR , anti-pERK , anti-ERK , anti-N-CAD , anti-p38MAPK , anti-pp38MAPK , anti-PG ((PG5.1) Progen #61005), anti-PKP2 , anti-phospho-Thr  and anti-α-tubulin ((DM1A), abcam, #7291) were used as primary antibodies. HRP-coupled secondary antibodies  and SuperSignal™ West Pico PLUS Chemiluminescent Substrate  were used to develop membranes with an iBright™ FL1500 developer (Thermo Fisher). NoStain™ Protein Labelling Reagent  was used as loading control. Quantification was performed using the iBright™ Analysis Software (Invitrogen).2. After replacing the enzyme mix by HBSS once the monolayer was detached, MTT was added for better visibility and monolayers were subjected to mechanical stress by shaking at 1.31 g for 5 min on an orbital shaker . The higher the amount of the monolayer fragmentation after mechanical stress, the lower the cellular cohesion and vice versa. Murine cardiac slice dissociation assays were performed similarly; however, Liberase-DH and dispase II were added simultaneously for 30 min. After adding MTT, equal mechanical stress using an electrical pipette was applied, and the resulting mix with heart tissue fragments, single dissociated cardiomyocytes and cell debris was filtered using a 70 µm nylon membrane . Pictures of the filtered cell suspension well were taken in gridded 96 well plates and stitched together using the AutoStich software , followed by dispase II  and incubation at 37°C, 5% COsoftware . The numCells were seeded on coverslips, fixed with 2% paraformaldehyde and permeabilized with 0.1% Triton X-100. After blocking with bovine serum albumin and normal goat serum, primary antibodies were added overnight at 4°C in a wet chamber. anti-DP , anti-DSG2  and anti-N-CAD  were used at 1:100 dilution. Fluorophore-coupled, species-matched secondary antibodies were added together with Phalloidin and DAPI . For image acquisition, a Leica SP5 II confocal microscope  with a 63X oil objective and the LAS-AF software was used. Z-scans were performed at 0.25 µm thickness spanning the whole cell volume. Image analysis was performed using the ImageJ software. For colocalization analysis, a region of interest at the membranes was chosen and the amount of stained pixels in this area in the other channels was calculated .1.5 mg of protein lysate was incubated with 1 µg of anti-DSG2/ADAM17 or control mouse IgG  or rabbit IgG  overnight at 4°C on a spinning wheel. Protein G Dynabeads™  were used to pull down the complexes by adding the lysate-antibody mix to prewashed beads for 1 h at 4°C. Afterwards, beads were isolated and washed using a magnetic rack, by washing twice with wash buffer 1 , thrice with wash buffer 2 , and twice with 1% Triton X-100 in PBS. After resuspension in Laemmli buffer, the samples were denatured for 10 min at 95°C and the lysate was separated from the beads using a magnet and loaded on a SDS-PAGE gel for Western blot analyses.via Western blot.Sulfo-ethylene glycol bis(sulfosuccinimidyl succinate) , a membrane-impermeable cross-linker, was used to analyze oligomerization of DSG2 at the cell membrane. To that end, after 90 min of TAPI-1 treatment, HL-1 cardiomyocytes were washed with ice-cold PBS. Then, 2 mM Sulfo-EGS in PBS was added for 30 min at room temperature. The reaction was stopped with TBS and cells were scraped into SDS lysis buffer and analyzed t-tests after outlier removal. Graphs are represented as mean ± standard deviation (SD). Data was normalized to the average control value in Western blots and dissociation assays in HL-1 cardiomyocytes. When analyzing dissociation assays performed in murine cardiac slices, normalization was performed to the respective control slice of the same mouse. Significance was assumed for p ≤ 0.05.Adobe Photoshop CS5 and ImageJ softwares were used for image processing. For Western blot quantification, the iBright™ Analysis Software was used. GraphPad Prism 8 was used for statistical analysis of the data, using unpaired Student’s Jup+/+ mice as well as in the Jup−/− murine AC model. ADAM17 protein levels were variable, though not significantly increased in Jup−/− mice. Phosphorylation of p38MAPK was increased in Jup−/− mice as compared to Jup+/+ controls  or vehicle treated HL-1 cardiomyocytes, respectively  proteins and Triton X-100 soluble  proteins . Howevernockdown .Since we observed an increase in DSG2 localization at the membrane, we next assessed whether DP, which provides the cytoskeletal anchorage to desmosomes, was affected by inhibition of ADAM17. Indeed, we found an increase in DP localization at the cell borders after inhibition of ADAM17 .Together, these data suggests that acute ADAM17 inhibition enhances the localization of DSG2 and DP at the cell borders and that DSG2 is a key driver of positive adhesiotropy upon ADAM17 inhibition.Next, we assessed whether increased DP and DSG2 localization at the membrane was paralleled by changes in the desmosomal complex. Therefore, we immunoprecipitated DSG2 and assessed the levels of other proteins of the desmosome which are in complex with DSG2. Immunoprecipitation of DSG2 revealed no changes in the desmosomal complex upon ADAM17 inhibition .2+-switch assay. During a Ca2+-switch, cells are depleted of Ca2+ by treating with the Ca2+-chelator EGTA for 90 min. The Ca2+-depletion leads to a disruption of desmosomes, since desmosomal contacts are Ca2+-dependent. After Ca2+-repletion, desmosomes are reassembled, and upon addition of mediators, their effect on the desmosomal assembly can be assessed. Ca2+-switch assays did not reveal any alterations in cellular cohesion upon inhibition of ADAM17, indicating that acute ADAM17 inhibition does not lead to enhanced desmosomal assembly . We therefore performed EGS linking experiments. We observed a trend of increased DSG2 oligomer formation at the cell membrane after ADAM17 inhibition, however, this trend was not significant . Thus, it is possible that the remaining EGFR protein levels were sufficient to mediate the TAPI-1 effect. Nevertheless, EGFR signaling was not affected by TAPI-1 treatment, suggesting that EGFR might not be involved in ADAM17 inhibition-mediated positive adhesiotropy. A recent study in keratinocytes also revealed that ADAM17 inhibition did not alter EGFR signaling  inflammation is involved in the pathogenesis of AC  ADAMis of AC  and c) ois of AC , acute i


Adverse local tissue reactions have been problematic as an implant-related complication in total hip arthroplasty (THA). Despite the absence of significant metal wear and corrosion, granulomatous pseudotumor has been reported to be caused by polyethylene wear. We performed a long-term follow-up study investigating the relationship between polyethylene wear and pseudotumor formation in THA.This study included 57 patients (64 hips) that underwent primary THA with metal-on-polyethylene or ceramic-on-polyethylene bearing over a minimum follow-up of 15 years. They were stratified into pseudotumor and non-pseudotumor groups and their linear wear rates of polyethylene liner and serum cobalt (Co) and chromium (Cr) ion levels were compared. Pseudotumor was diagnosed on metal artifact reduction sequence-MRI according to its composition and wall thickness using the Hauptfleisch classification.P < 0.001). According to an analysis of the receiver operating characteristic curves, the cutoff level of the wear rate to discriminate between pseudotumor and non-pseudotumor patients at 15 years was 0.11 mm/year .The incidence of pseudotumor was 34% (22/64 hips) at the mean follow-up of 16.9 years. Metal ion levels did not differ between the pseudotumor and non-pseudotumor groups, and none of the patients exceeded the Co/Cr ratio of 2.0 μg/L. Moreover, the wear rate in the pseudotumor group was 1.8 times greater than in the non-pseudotumor group (0.14 vs. 0.08 mm/year, Our results might provide new insights into excessive polyethylene wear potentially leading to the future development of both pseudotumor and osteolysis. Further studies are needed to clarify the direct relationship between polyethylene wear and pseudotumor and the mutual effects of osteolysis and pseudotumor in particle reactions. Periprosthetic osteolysis and aseptic loosening have been one of the most common reasons for revision in total hip arthroplasty (THA), which commonly proceed via foreign body reactions to wear particles of ultra-high molecular weight polyethylene  bearing . Their rOn the other hand, adverse local tissue reactions (ALTR) and pseudotumor formation are also problematic as implant-related complications in THA. In recent publications, ALTR has been associated with mechanical wear and corrosion of metallic components at the modular head-stem taper junction or related to modular stems , 5. HoweIn the above context, we designed the long-term follow-up study to investigate the following: (1) the incidence of pseudotumor formation after primary THA using polyethylene; and (2) the best cutoff threshold of the wear rate in differentiating between pseudotumor and non-pseudotumor patients. This is the first report on the relationship between polyethylene wear and pseudotumor formation in THA.The institutional review board of our hospital approved this retrospective cohort study. Between April 1998 and December 2003, primary cementless THAs were performed for 89 patients (98 hips) at a single institution. All patients received a cementless plasma-sprayed porous-coated RingLoc® acetabular shell, a 30-kGy cross-linked ArCom® polyethylene liner, a 28 mm cobalt chromium (CoCr) or zirconia femoral head and a single design of proximally porous-coated Bi-Metric® femoral stem. Zimmer-Biomet, Warsaw, IN (formerly known as Biomet Inc.) supplied all components. All operations were performed by three experienced hip surgeons  via posterior approach under general anesthesia. Among the 89 patients, 6 patients (6 hips) died for reasons unrelated to THA. The analysis excluded 8 patients (10 hips) who had been followed for less than 15 years. Moreover, 4 patients (4 hips) with rheumatoid arthritis and 14 patients (14 hips) with incomplete MRI data were excluded. A total of 57 patients (64 hips) were included in the analysis and were followed up for a mean of 18.5 years. Demographic data and clinical information are shown in Table Harris hip scores (HHS) were used to assess clinical outcomes before and after THA. Standard anteroposterior (AP) pelvic and lateral radiographs of hips were obtained at the follow-up to evaluate periprosthetic osteolysis and aseptic loosening of femoral stems and acetabular cups in each of the 7 Gruen zones  and the Two-dimensional polyethylene liner wear  and acetabular cup inclination and anteversion angle were measured on AP-pelvic radiographs taken in the supine position using Martell’s Hip Analysis Suite software , which is a computer-assisted semiautomated edge detection system. Liner head penetration rates (mm/year [y]) were measured radiographically for all THAs at 5, 10 and 15 years after surgery. Annual radiographs after revision surgery were excluded. All radiographic measurements were analyzed by the same independent observer (T.I.).Metal artifact reduction sequence magnetic resonance imaging (MARS-MRI) was performed after a minimum of 15 years using a 1.5-T scanner  to diagnose a periprosthetic pseudotumor formation regardless of patients’ symptoms. All MARS-MRIs were independently reviewed by a senior radiologist familiar with pseudotumor diagnosis. Pseudotumors were classified according to their composition and wall thickness using the Hauptfleisch classification : Type I,Serum metal  ion levels and the Co/Cr ratio were measured 1 year after MARS-MRI. Serum Co levels in patients were measured using inductively coupled plasma mass spectrometry at Mayo Medical Laboratories , while Cr levels were using atomic absorption spectrometry at LSI Medience Corporation . Both Co and Cr ions had detection limits of 0.1 μg/L.U test and the χ2 test were used to compare the patient groups with and without pseudotumor. The r and Cramer’s V were reported as indicators of effect sizes for the Mann–Whitney U and χ2 test, respectively, considering that r and V = 0.1 represent a small effect, r and V = 0.3 represent a medium effect, and r and V = 0.5 represent a large effect [P value of < 0.05 was considered statistically significant.The Mann–Whitney's e effect , 19. To P < 0.001). Acetabular and femoral osteolysis were observed in 15 hips (23%) and 14 hips (22%), respectively  who had MoP (34 hips) and ceramic-on-polyethylene (CoP: 30 hips) bearings. The mean follow-up was 18.5 years (range: 15–23 years). At the time of final follow-up, the mean  HHS had increased significantly from 43.3 (4.7) to 87.2 (5.9) (3 (1.3–9.8). Moreover, five of 22 hips (23%) were symptomatic, with a median (IQR) volume of 11.4 cm3 (3.5–15.9), and 4 of the symptomatic 5 hips underwent revision surgery after a mean (SD) follow-up period of 12.4 (2.5) years (Table P = 0.061). The intraoperative histopathological findings of the pseudotumors revealed foreign body reactions to large polyethylene wear particles with 20–1000 μm, with no histological evidence of corrosion at the head-neck interfaces.The mean (SD) time elapsed between the primary THA and MARS-MRI scanning was 16.9 (1.8) years. MARS-MRI revealed the presence of pseudotumor formation in 22 hips (34%). The pseudotumor was classified as type I , type II , and type III  , preoperative hip extension limitation , frequency of acetabular osteolysis , PT at 15 years , changes in cup inclination , change in cup anteversion , and change in PT  between 5 and 15 years. The median (IQR) wear increased over time from 0.46 (0.37–0.59) mm at 1 year to 1.20 (0.85–1.39) mm at 15 years in patients without pseudotumor, while it increased from 0.53 (0.46–0.80) mm at 1 year to 2.02 (1.74–2.34) mm at 15 years in those with pseudotumor , 10 , and 15 years  . On the other hand, no significances between the groups were found regarding body mass index (BMI), head materials, cup diameter, polyethylene liner thickness, HHS, cup orientation at 5 and 15 years, frequency of femoral osteolysis and metal ion levels  of wear rates determined at 5, 10 and 15 years with AUC values of 82, 89 and 91%, respectively. The most sensitive and specific cutoff values of wear rates at 5, 10 and 15 years were 0.23 , 0.12 , and 0.11 mm/y , respectively.Figure Se. = 100%, and Sp. = 90%) in diagnosing ALTR associated with corrosion in MoP THA when the optimal cutoff of Co ion level was set at 1.0 μg/L. Kwon et al. [P < 0.001, r = 0.65, Table The most common failure mode related to tribocorrosion in hard-on-soft THA is mechanically-assisted crevice corrosion (MACC), which has been observed particularly when a large diameter head was used –24. In mn et al.  suggesten et al. . In the P = 0.033, Table e.g., age, gender, vertebral fractures, preoperative anterior PT, spinal degeneration, and history of spinal fusion surgery) [It is important to note that factors associated with an increased/decreased polyethylene wear rate include patient-related factors, surgeon-related factors, and implant-related factors. Furthermore, common wear-related factors have been identified as gender, age, BMI, activity level, head diameter, head material, liner thickness, cup diameter, cup alignment and pelvic change , 29, 30.surgery) . A detaiThe cellular response to polyethylene wear debris might be affected not only by the number of particles but also their size, morphology, and distribution. The cellular response to different sizes of polyethylene wear particles has previously been studied. Howie et al.  demonstrLarge polyethylene particles are generated commonly because of abnormal wear attributed to excessive mechanical loading such as dislocation, subluxation, impingement, and edge loading , 36. TheP > 0.999) . This wear threshold for pseudotumor is higher than that to increase the frequency of osteolysis reported previously in the range of 0.05 [P = 0.028). In the above contexts, acetabular osteolysis appears to induce the development of pseudotumor.At the mean follow-up of 16.9 years, the incidence of pseudotumor formation in our patients was 34% 22/64 hips). Although there was no significant difference in its incidence between the head types, 35% (12/34 hips) of MoP patients and 33% (10/30 hips) of CoP patients experienced the development of pseudotumor ( of 0.05  to 0.1 m2/64 hips of 0.05 –42 hypotThis study had some limitations. First, it was retrospective study with a relatively small sample size and the two cohorts were not randomized. However, large effect sizes were obtained for all significant comparisons in the wear rates, suggesting a strong degree of practical significance for the primary results Table . TherefoDespite the aforementioned limitations, our results might provide new insights into excessive polyethylene wear potentially leading to the future development of both pseudotumor and osteolysis. Further studies are needed to clarify the direct relationship between polyethylene wear and pseudotumor and the mutual effects of osteolysis and pseudotumor in particle reactions.


Zinc finger and bric-a-brac/tramtrack/broad (ZBTB) domain-containing proteins have been reported to be associated with many tumors’ development. However, in tumor initiation and progression, the role of ZBTB9, one of the protein family, and its prognostic value were yet to be elucidated in Liver Hepatocellular Carcinoma (LIHC).We used R software and online bioinformatics analysis tools such as GEPIA2, cBioPortal, TIMER2, Metascape, UALCAN, STRING, TISIDB, and COSMIC to investigate ZBTB9’s characteristics and function in LIHC, including abnormal expression, carcinogenic role, related signaling pathways and prognostic value. Furthermore, cell experiments  and analyses based on clinical samples (such as immunohistochemistry (IHC) and promoter methylation analysis) were conducted to verify pivotal conclusions.ZBTB9 was overexpressed in LIHC samples compared to adjacent normal tissues. Through the analysis of genomic alteration and promoter hypomethylation, the clinical value and etiology of abnormal expression of ZBTB9 were preliminarily exlpored. Subsequent evidence showed that it could result in tumor progression and poor prognosis via activating cell cycle, DNA repair, MYC, and KRAS-associated signaling pathways as well as rendering immune dysregulation. After the knockdown of ZBTB9, evidently inhibited capacities of tumor cells proliferation and migration were observed. These results together indicated that ZBTB9 could be a promising prognostic biomarker and had the potential value to offer novel therapeutic targets for LIHC treatment.ZBTB9 was identified as a novel biomarker to predict the prognosis and tumor progression in LIHC, and a promising therapeutic target to invert tumor development.The online version contains supplementary material available at 10.1186/s12967-022-03790-0. Liver Hepatocellular carcinoma (LIHC) is one of the most aggressive cancers. Cancer statistics showed 906,000 new liver cancer cases and 830,000 deaths worldwide in 2020, and the type of 75%–85% liver cancer cases was LIHC –3. In thRecently, increasing attention has been paid to ZBTB proteins, which are the family proteins of nuclear transcription factors . These gZBTB9 is a member of ZBTB proteins, and the relationship between its expression and tumor development is poorly studied. In the results of preliminary bioinformatic analysis, we found it was overexpressed in LIHC compared to adjacent normal tissues. Based on this, we conducted further analyses and necessary experiments to investigate its characteristics and prognostic value of it in LIHC. In conclusion, the present study aims to confirm the potential capacity of ZBTB9 to be a novel biomarker and therapeutic target, and simultaneously, to provide novel insight into the underlying molecular mechanisms of LIHC.http://timer.cistrome.org/) [https://www.ncbi.nlm.nih.gov/) database.Tumor Immune Estimation Resource 2.0 (TIMER2) (me.org/)  online thttp://xena.ucsc.edu/) [The gene expression arrays and clinical information of the Cancer Genome Atlas (TCGA)  LIHC cohsc.edu/)  databasesc.edu/) .http://gepia2.cancer-pku.cn/) [By the tool of Gene Expression Profiling Interactive Analysis 2.0 (GEPIA2) (pku.cn/) , we acquhttps://cn.string-db.org/) was a powerful online tool for bioinformatic analysis which could analyze the protein–protein interaction network, based on the evidence from experiments and consensus.STRING . H-SCORE (H-SCORE = ∑(pi x i) = (percentage of weak intensity × 1) + (percentage of moderate intensity × 2) + (percentage of strong intensity × 3) –17) was With the pROC package of R (v 3.6.3), the receiver operating characteristic curve (ROC) was depicted to show its diagnostic value.The Kaplan–Meier survival analysis and cox regression model were performed via the “survminer” and “survival (v 3.2-7)” packages of R, and the P-value, 95% confidence intervals (CI), and hazard ratio (HR) were also computed.https://www.cbioportal.org/) [cBioPortal database  , 21 was http://www.sanger.ac.uk/cosmic/) [The Catalogue of Somatic Mutations (COSMIC) (cosmic/)  is anothhttp://ualcan.path.uab.edu/analysisprot.html) [Through UALCAN (ot.html) , we acquMSP analysis was performed using primers for methylated or unmethylated DNA designed using MethPrimer. Briefly, 2 μl of bisulfite-treated genomic DNA was amplified using TaKaRa EpiTaq HS (Takara Bio). The cycles setting is 95 °C for 5 min, followed by 35 cycles of 94 °C for 20 s, 60 °C for 30 s, and 72 °C for 20 s, followed by a single cycle of 72 °C for 5 min. then, the PCR products were analyzed using 2% agarose gel electrophoresis. The results were quantitated via ImageJ software . All meaZBTB9-M-F: 5′-GGTGTTATATTTAATTATCGGGAAAC-3′ZBTB9-M-R: 5′-AACTACTACAAACGAAAAAAACGAT-3′ZBTB9-UM-F: 5′-GTTATATTTAATTATTGGGAAATGT-3′ZBTB9-UM-R: 5′-AACTACTACAAACAAAAAAAACAAT-3′.https://discover.nci.nih.gov/cellminer/home.do) [We logged in CellMiner online database (home.do) , 25 to ghttp://cis.Hku.hk/TISIDB/) [By the TISIDB database (TISIDB/) , we analIHC of ZBTB9 , CD8A , and FOXP3  proteins in the 20 samples containing 10 tumor and 10 adjacent normal tissues was conducted to explore the relationship between ZBTB9 and TIME.The Gene set enrichment analysis (GSEA)  method whttp://metascape.org) tool [Gene Ontology (GO) and Kyoto Encyclopedia of Gene and Genomes (KEGG) pathway analysis were utilized to investigate the function of the gene set with R package clusterProfiler (3.14.3) and org.Hs.eg.db (3.10.0). GO analysis is a common method used for annotating genes and gene products and for identifying molecular function (MF), biological process (BP), and cellular components (CC). KEGG is a collection of databases for systematic analysis of gene functions and associating related gene sets with their pathways. In addition, we also utilized the Metascape (rg) tool  to analyhttps://www.editorialmanager.com/jtrm/default1.aspx) [With GSCALite (t1.aspx) , the assHepG2 cells were obtained from the Chinese Academy of Sciences Cell Bank and cultured in Dulbecco’s modified Eagle’s medium (DMEM) , supplemented with 10% fetal calf serum (FBS) , 100 U/ml penicillin, and 100 μg/ml streptomycin , and cultured at 37 °C in a 5% CO2 humid atmosphere.To block ZBTB9 expression in HepG2 cells, two small interferings (si) RNAs against ZBTB9 (siZBTB9-1 and siZBTB9-2) and one negative control (siCtrl) RNA were designed and synthesized by Sangon Biotech Co., Ltd. For transfection, cells were seeded into 6-well plates at a concentration of 1 × 10^5 cells/well and maintained at 37 °C until 70% confluent. Then, cells were transfected with siRNAs or siCtrl using Lipofectamine® 3000  following the manufacturer’s protocols. The antisense sequences of the three siRNAs were shown as followed:siZBTB9-1: 5′-TTTCTGAGCACTGATCTACTA-3′.siZBTB9-2: 5′-TAACACTGCTTTATGAGCCCT-3′.siCtrl: 5′-TTCTCCGAACGTGTCACGTTT-3′.Relative expression of ZBTB9 in HepG2 cells was evaluated via RT-qPCR. We extracted total RNA from cells using TRIzol reagent . cDNA was synthesized using the RevertAid First Strand cDNA Synthesis kit  following the manufacturer's instructions. RT-qPCR was conducted using the iQ™ SYBR Green Supermix . The following primers were used for qPCR:ZBTB9-F: 5′-TCGGCGGGAAGGACAATC-3′,ZBTB9-R: 5′-TAGACTCCACGCGGGATGAA-3′,GAPDH-F: 5′-ACAACTTTGGTATCGTGGAAGG-3′GAPDH-R: 5′-GCCATCACGCCACAGTTTC-3′.The 2-ΔΔCt method was conducted to calculate the relative expression levels of targets.For WB assay, the molecular weight of ZBTB9 and GAPDH are 50.602 kDa and 36.053 kDa, respectively, and total proteins were extracted from HepG2 cells using RIPA lysis buffer and subsequently were quantified using the Bradford method . Twenty micrograms of protein were loaded and separated by polyacrylamide gel electrophoresis and then transferred to PVDF membranes (Bio-Rad). After blocking with 5% BSA, membranes were incubated with specific antibodies for ZBTB9 , and GAPDH  at 4 °C overnight. On the next day, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody (1:3000) for 2 h at room temperature. The Quantity One software package  was used for the quantitation of signal intensities.HepG2 cells were seeded into a 6-well plate. When the cells' confluence reached 80%, we scratched the monolayer cells with a sterile micropipette tip. The floating cells were then washed with PBS. Wound healing within the scrape line was observed at 0 h and 16 h. Triplicate wells for each condition were examined.Flow cytometry analysis was performed to estimate the effects of ZBTB9 knockdown on the HepG2 cell cycle. After 48 h transfection with siZBTB9 or siCtrl, HepG2 cells were trypsinized and centrifuged for 10 min at 2000 rpm, followed by three washes with pre-cold PBS. Then, cells were fixed with 70% ethanol at -20˚C overnight, and cell cycle distribution was analyzed using the cell cycle kit (BestBio) and a Beckman Coulter FACSCalibur flow cytometer .The effects of ZBTB9 knockdown on LIHC cell proliferation were assessed by colony formation assay. After 48 h transfection with siZBTB9 or siCtrl, HepG2 cells were seeded into 6-well plates at a density of 1000 cells/well, followed by 1 week of incubation at 37 °C with 5% CO2. Subsequently, colonies were fixed using methanol for 1 h and stained with Giemsa solution for 20 min. The number of colonies (> 50 cells) was manually calculated under a microscope, and triplicate wells were evaluated for each group.Transwell assay using 24-well plates with an 8-µm pore chamber , and Matrigel matrix was diluted 1:8 to coat the upper side of the membrane at the bottom of the transwell chamber. Cells were first cultured in 2% FBS-DMEM medium for 12 h for starvation treatment and suspended in FBS-free DMEM medium, then were added to the upper chamber (1 × 10^5 cells/well), respectively. Meanwhile, DEME medium with 10% FBS was added to the lower compartment. The plates were incubated for 72 h in the incubator. After incubation, cells that migrated to the lower surface of the filter membrane were fixed with 4% paraformaldehyde and stained with 0.5% crystal violet. Cells remaining on the upper surface of the filter membrane were gently scraped off with a cotton swab. The lower surfaces were captured by the inverted microscope, and the counting was repeated three times.Parts of packages of R were introduced as previously reported, ggplot2 package and Graphpad prism 9.0 software was utilized to visualize the results of statistical analyses, besides, the chi-squared test, Student’s test, Wilcoxon test, and Kruskal–Wallis tests were used appropriately to calculate the significance of differences in data between groups.Genes with aberrant expression in tumor samples were considered to probably participate in tumor initiation and development. Herein, we primarily found that ZBTB9 was significantly upregulated in 15 types of tumors compared to corresponding normal tissues with TIMER2 pan-cancer data, including BLCA, BRCA, CHOL, COAD, ESCA, GBM, HNSC, HNSC-HPV (−), LIHC, LUAD, LUSC, PRAD, READ, STAD and UCEC   analysis based on LIHC cohorts  were perFurthermore, with the coxph function of the survival package, cox proportional hazard regression analysis was rendered to analyze the HR of ZBTB9 among cancers (only the significant results were given), which indicated that ZBTB9 was a significant risk factor in many cancers, including GBMLGG , SKCM-M , LAML , SARC , SKCM , LAML  and LIHC  , pathologic stage (P < 0.001), T stage (P < 0.001) M stage (P = 0.017), and tumor status (P < 0.001) were all significantly correlated with OS. Besides, ZBTB9 expression was also significantly correlated with OS in multivariate cox regression analysis P = 0.014), suggesting that ZBTB9 was an independent prognostic factor in LIHC patients , N stage: N0 (HR = 1.63), M stage: M0 (HR = 1.80), pathologic stage: Stage III (HR = 2.59), tumor status: With tumor (HR = 1.76), gender: male (HR = 1.98), race: Asian (HR = 1.99), Race: white (HR = 1.89), age: ≤ 60 (HR = 1.87), age: ≥ 60 (HR = 1.81), BMI: ≤ 25 (HR = 2.06), residual tumor: R0 (HR = 1.84), histologic grade: G1 (HR = 5.82), histologic grade: G2 (HR = 1.44), histologic grade: G3 (HR = 2.08), adjacent hepatic tissue inflammation: None (HR = 3.01), AFP: ≤ 400 ng/ml (HR = 2.33), Albumin: ≥ 3.5 g/dl (HR = 1.93), Prothrombin time: ≤ 4 (HR = 1.81), Prothrombin time: > 4 (HR = 2.19), Child–Pugh grade: A (HR = 1.75), Fibrosis ishak score: 0 (HR = 3.37), Vascular invasion: Yes (HR = 2.15), with all P values < 0.05 , G2M checkpoint , MYC TARGETS V1 , EPITHELIAL MESENCHYMAL TRANSITION , KRAS SIGNALING DN ,  and ZBTB9 gene expression level was analyzed, which showed that ZBTB9 level was significantly negatively related to the IC50 of BMS-911543 , Defactinib , ITRI-260 , Dovitinib , BPR1J-097 , TPX-0005 , CG-806 , CFI-402257  and BLU-667 , and positively related with Fludarabine , Cladribine  , KCTD20 , NFYA , RPL7L1 , SLC39A7 , TAF11 , WDR46   [With GSCALite (t1.aspx)  tool, weGiven the above multi-aspects findings, ZBTB9 was an oncogene driving tumor initiation and progression in LIHC, which could lead to poor prognosis via activating cell proliferation and rendering immune dysfunction. We then performed a series of experiments to verify whether the malignant biological behaviors could be inhibited via the knockdown of ZBTB9 at the cell level.Firstly, the knockdown efficiency of siZBTB9-1 and siZBTB9-2 was evaluated by RT-qPCR, which showed that the siRNAs reduced the mRNA level of ZBTB9 in HepG2 cells Fig. A, and thThe effects of ZBTB9 knockdown on cell proliferation were examined via colony formation assay. Compared to the siCtrl group, the number of HepG2 colonies groups was significantly reduced in the ZBTB9 knockdown groups Fig. E, F.Flow cytometry analysis was performed to analyze the impacts of ZBTB9 knockdown on the cell cycle. The results suggested that the cell number at the G1 phase of the two ZBTB9 knockdown groups was significantly increased compared to the siCtrl group, and the cell number in the S and G2 phases was decreased , and two LIHC cohorts’ data from GEO also proved its abnormal expression. In addition, the levels of ZBTB9 showed the relation with higher tumor grade and individual stages for LIHC patients. Given promoter hypomethylation of oncogenes has been considered as a tumor initiation and development promotive factor, it could result in gene overexpression, and downstream signaling pathways activation , 33, ourSurvival analysis was conducted to evaluate the prognostic value of ZBTB9, which indicated that LIHC patients who had high ZBTB9 expression tended to possess shorter OS  and DSS  based on TCGA data. Similarly, the survival analysis results of the tissue microarray further confirmed that ZBTB9 was also an evidently risk factor for LIHC patients OS. In addition, the Cox regression analysis based on pan-cancer data also demonstrated that ZBTB9 was a notable risk factor in many cancers, including LIHC. These findings all indicated that ZBTB9 was not only an overexpressed gene but a significant risk factor for the prognosis of LIHC patients. Genomic alteration and promoter hypomethylation could be the etiology for its abnormal expression. Furthermore, the correlation analysis between the efficacy of clinical drugs and ZBTB9 expression showed that there were 9 in 11 significantly negative correlations with drug efficacy , suggesting the value of ZBTB9 in the guidance for LIHC patients’ precise treatments.Given the above findings, we conducted a series of analyses to investigate the downstream mechanisms for its carcinogenetic and risk role. It has been reported that the characteristics of TIME play a crucial role in LIHC initiation and progression , 35, theFurther GSEA analysis showed that in high ZBTB9 expression samples, the G2M checkpoint, epithelial-mesenchymal transition, E2F targets, and KRAS-related signaling pathways were significantly activated, which has been reported to participate in tumor proliferation and progression , 37. In Similarly, based on ZBTB9-related genes, the results of correlated biological functions and signaling pathways analysis indicated that these genes participated in triggering cell cycle activation, which suggested that these genes possessed synergy effects on the promotion of tumor initiation and development at some extent.To credibly identify the role of ZBTB9 in LIHC, we further conducted a series of cell experiments. After ZBTB9 knockdown, the cell cycles of HepG2 cells were mainly stuck in the G1 phase with P < 0.001 and the invasion assay showed that after the suppression of ZBTB9, the cell competence of migration was significantly impaired (P < 0.001). In addition, after the same time interval, wound healing assay and colony assay indicated that a wider wound and a less number of colonies were detected in the ZBTB9 knockdown groups compared to siCtrl groups. Which confirmed the promotive role of ZBTB9 in tumor cell development.It needs to be acknowledged that there are limitations of our study. The clear downstream molecule of ZBTB9 expression was not comprehensively identified with current experiments and the limited number of LIHC samples. More rigorous in vivo experiments also need to be conducted to confirm the current findings and help to reveal novel characteristics for ZBTB9 role in LIHC, and it is also what we will further explore in the future.This study is based on the evidence from multi-omics, experiments, and clinical samples to identify that ZBTB9, a member of ZBTB proteins, could promote tumor proliferation and migration, which possesses the strong potential to be a novel biomarker and to offer new treatment targets for LIHC patients.Additional file 1: Figure S1. Results analysis of the MSP agarose gel electrophoresis. M labels represent the results of promoter methylation, and U labels represent the results of non-promoter methylation. The odd labels represent adjacent normal tissues and even labels represent tumor tissues (A). The red dots and blues dots represent the quantitative results of tumor and adjacent normal tissues, and the green dots in the right part represent the difference value  between the quantitative results of paired adjacent normal tissues and tumor tissues, with pixels as scales. The result based on paired T-test showed that ZBTB9 promoter methylation was evidently lower in tumor tissues than the paired adjacent normal tissues .Additional file 2: Figure S2. Correlation between ZBTB9 level and CD8A, FOXP3 in TIME (50 μm). Tumor tissues were obtained from two LIHC patients, who had different expression level of ZBTB9, marked as sample #1 and sample #2. The blue dots represent cell nuclear, and the brown or yellow stainings represent the target proteins. According to the intensity and the comparison of the number of brown or yellow stainings and blue dots, the relatively quantitation of protein expression could be observed. In the relatively high ZBTB9 level patient (sample #2), the relatively low levels of CD8A (D) and FOXP3 (F) were observed.Additional file 3: Figure S3. The efficacy of siRNAs was detected via WB assay. The groups contained the samples of siCtrl, siZBTB9-1 and siZBTB9-2, and two target proteins, ZBTB9 and GAPDH. Results showed that the expression levels of ZBTB9 were significantly downregulated after inhibition.