Inhibition of amyloid-β peptide (Aβ) accumulation in the brain is a promising approach for treatment of Alzheimer’s disease (AD). Aβ is produced by β-secretase and γ-secretase in endosomes via sequential proteolysis of amyloid precursor protein (APP). Aβ and APP have a common feature to readily cluster to form multimers. Here, using multivalent peptide library screens, we identified a tetravalent peptide, LME-tet, which binds APP and Aβ via multivalent interactions. In cells, LME-tet-bound APP in the plasma membrane is transported to endosomes, blocking Aβ production through specific inhibition of β-cleavage, but not γ-cleavage. LME-tet further suppresses Aβ aggregation by blocking formation of the β-sheet conformation. Inhibitory effects are not observed with a monomeric peptide, emphasizing the significance of multivalent interactions for mediating these activities. Critically, LME-tet efficiently reduces Aβ levels in the brain of AD model mice, suggesting it may hold promise for treatment of AD. Multivalent peptide library screens identify a tetravalent peptide that binds APP and A β via multivalent interactions and reduces A β in the brains of Alzheimer’s disease model mice. In neuronal cells, Aβ is produced by the sequential proteolytic cleavage of amyloid precursor protein (APP), a type I transmembrane protein4. APP present in the plasma membrane is internalized into early endosomes and then into late/recycling endosomes, where it is cleaved by β-secretase to produce a soluble N-terminal fragment, sAPPβ, and a membrane-bound C-terminal fragment, CTFβ (C99)5. C99 is subsequently cleaved by γ-secretase to produce an APP intracellular domain and Aβ7. Alternatively, within the plasma membrane, APP can be cleaved by α-secretase to produce a soluble N-terminal fragment, sAPPα, and a membrane-bound C-terminal fragment, CTFα9. Aβ produced in cells is released into the extracellular space, where it aggregates, leading to the formation of amyloid plaques in the brain, the typical pathological feature of AD. Thus, compounds that inhibit production of intracellular Aβ and/or the aggregation of extracellular Aβ in the brain may be effective therapeutic agents against AD.Alzheimer’s disease (AD) is a progressive neurodegenerative disorder in humans, characterized by the presence of senile plaques within the brains of affected individuals, the main constituent of which is amyloid-β peptide (Aβ)11. β-secretase-deficient mice, in particular, exhibit a wide range of pathological symptoms, including axon guidance defects12 and seizures13, and even conditional β-secretase knock-out in adulthood impairs axon guidance14. These observations underscore the need for new therapeutic strategies based on substrate-specific inhibition. Compounds that bind to or modulate APP and as a result, specifically inhibit its β-cleavage, are particularly attractive, as this is the rate-limiting step for Aβ production15. However, inhibitors with this substrate-specific activity have not been developed.Inhibitors against β-secretase or γ-secretase are one such class of compounds that have been proposed as a possible approach for suppressing Aβ production. However, inhibition of these enzymes can lead to cleavage defects in a variety of other substrate proteins, resulting in severe side-effects16. Notably, of the various Aβ species processed by γ-secretase, Aβ42 has been shown to have a greater propensity to aggregate compared to Aβ40, the major processed form18. APP and C99 can also easily oligomerize to form homodimers through their extracellular domains and possibly through their juxtamembrane and transmembrane domains, as well21. Thus, a compound that tightly binds to APP, C99, and/or Aβ, particularly Aβ42, could represent an ideal therapeutic agent to inhibit production of Aβ by modulating APP metabolism in cells and/or blocking the subsequent extracellular oligomerization of Aβ.Aβ is prone to aggregate into β-sheet conformations, which are detected in the brains of patients with AD, first in the form of Aβ oligomers, and then as Aβ fibrils22, such as Shiga toxin26, a major virulence factor of enterohemorrhagic Escherichia coli, and influenza virus hemagglutinin27. Critically, we found that these tetravalent peptides, but not monomeric peptides with the same motifs, bind to their target molecules with high affinity and potently inhibit their pathogenic function. Thus, our findings clearly demonstrate the importance of multivalent interactions for inhibitors that effectively target molecules that form multi-subunit or oligomeric structures. This further suggests that an optimal inhibitor for APP, C99, and/or Aβ should also engage in multivalent interactions with its target(s).Previously, we developed a method to screen multivalent peptide libraries and identify tetravalent peptides with the ability to directly bind bioactive molecules that function in multi-subunit formsIn this study, by targeting the N-terminal region of Aβ (Aβ1–28), we developed a new method for identifying tetravalent peptides that bind this region via multivalent interactions. Using this method, which is based on an affinity-maturation screening of tetravalent peptide libraries synthesized on a cellulose membrane, we identified a series of peptides that tightly bind to Aβ1–28. We further show that one of these peptides, LME-tet, can bind to APP in cells and specifically inhibit the intracellular production of Aβ by blocking its β-cleavage and the subsequent extracellular release of Aβ. In addition, LME-tet can inhibit aggregation of Aβ42 with remarkable potency through direct binding to the Aβ42 and shows protective effects against Aβ accumulation within the brain in a mouse model of AD. Thus, LME-tet may represent a promising therapeutic candidate for treatment of AD in humans suffering from this devastating disease.To identify a compound that modulates APP metabolism through direct interaction with APP, C99, and/or Aβ and inhibits production of Aβ in cells, we targeted the N-terminal region of Aβ (Aβ1–28), which is conserved in all three proteins Fig. . We then28. Among all peptides tested, only LME-tet (containing the Pro-Lys-Leu-Arg-Met-Lys-Glu motif) was able to reduce the amount of extracellular Aβ to 59% of that in the control, although the intracellular C99 and Aβ levels were not significantly affected  times higher than that of intracellular Aβ after incubation of 7WD10 cells for 48 h  cells stably expressing human APPted Fig. . The tot29. The relatively higher binding affinity to Aβ42 vs. Aβ40 may thus reflect its propensity to aggregate, thus exaggerating the clustering effect.To further investigate the significance of multivalent interaction between LME-tet and its putative target molecules, the direct binding of LME-tet to Aβ40, Aβ42, and Aβ1–28 was measured using enzyme-linked immunosorbent assays (ELISAs). We found that LME-tet, but not LME-mono, efficiently binds to Aβ1–28, Aβ40 (apparent Kd = 2.7 μM), and Aβ42 (apparent Kd = 0.20 μM) Fig. . NotablyTo determine whether LME-tet binds directly to APP, C99, and/or Aβ in cells, we performed coprecipitation assays. To this end, 7WD10 cells were treated with biotinylated LME-tet or LME-mono for 30 min at 37 °C. Cells were then lysed, and proteins that coprecipitated with each peptide were analyzed by western blot. Results show that APP, C99, and Aβ coprecipitated with LME-tet, but not LME-mono Fig. , indicatWe then examined whether LME-tet stimulates lysosomal degradation and/or autophagy to inhibit the Aβ production, by staining for lysosome-associated membrane glycoprotein 1 (LAMP1) or with Lysotracker, which are makers for the lysosome and acidic compartment, respectively. Our results indicate that 1 h after endocytosis of APP, only faint co-localization between each marker and APP is observed, and colocalization is not further increased in response to complex formation between APP and LME-tet Fig. . We furtBecause the endosome is the primary site for APP processing, we next determined the effects of LME-tet on cleavage of APP by α-secretase and β-secretase. To this end, 7WD10 cells were treated with LME-tet for 48 h, and levels of intracellular APP, as wells extracellular sAPPα and sAPPβ, which are produced by α- and β-secretase, respectively, were measured by western blot. We found that the total amounts of glycosylated APP (mature form) and the immature form were increased by 1.6 and 1.3 times, respectively, by LME-tet treatment Fig. . Further30. We therefore treated 7WD10 cells stably expressing St6gal1  with LME-tet for 48 h and measured the amounts of extracellular St6gal1 fragment. Results show that production of extracellular St6gal1 fragment is not affected by LME-tet treatment but is completely suppressed by a β-secretase inhibitor II, which directly inhibits the catalytic activity of this enzyme  with LME-tet for 48 h does not affect the production of NICD, whereas the γ-secretase inhibitor DAPT efficiently inhibits NICD production , a representative intracellular β-secretase substrate, is known to be cleaved by β-secretase to produce a St6gal1 fragment, which is released from cellsTo further confirm the specificity of the inhibitory effect of LME-tet on APP, we measured the direct effects of LME-tet on β- and γ-secretase activities in vitro. For assessing β-secretase activity, we measured production of β-cleaved C-terminal fragment (Aβ33-FLAG) from a FLAG-tagged fragment of human APP (residues 633–685 in APP751), which contains the Aβ1−28 region. We found that LME-tet, but not LME-mono, efficiently inhibits production of Aβ33-FLAG  analysis, we found that LME-tet markedly delays the conformational change of Aβ42, characterized by the formation of β-sheet structures, which is a hallmark of Aβ42 oligomerization Fig. . In cont33. We examined the protective effect of LME-tet on the cytotoxic activity of Aβ42 prefibrillar aggregates using the Neuro-2a neuronal cell line. Incubation of the cells with Aβ42 for 48 h in the absence of LME-tet reduced cell viability by 20%. The presence of LME-tet substantially rescued the viability by 5% , which carries the Swedish (NL), Beyreuther/Iberian (F), and Arctic (G) mutations (AppNL-G-F/NL-G-F mice). These mice show accumulation of Aβ in the brain and display pathological features of AD32. For these experiments, to increase stability of LME-tet in mice, we used a protease-resistant N-terminal acetylated form of LME-tet (Ac-LME-tet), which shows a similar inhibitory effect on Aβ production in 7WD10 cells , although LME-tet does not enhance this process to inhibit Aβ production 42 and β-galactoside α2, 6-sialyltransferase44. Notably, sterol-linked β-secretase inhibitors, which target endosomal β-secretase, have been shown to block cleavage of APP, but not non-amyloid substrates, such as NRG or the axon guidance molecule L146. Use of such hybrid inhibitors, however, cannot exclude the possibility that they may inhibit β-cleavage of other non-amyloid substrates, as they still target the β-secretase enzyme. In contrast, by targeting the ectodomain of C99, rather than β-secretase or γ-secretase itself, we previously identified a set of monomer peptides with the CDCYCXXXXCXCXSC motif that inhibit β- and γ-cleavage by binding to APP and C99, respectively, and thus inhibit Aβ production in cells47. Interestingly, here we found that LME-tet, which also targets the ectodomain, inhibits Aβ production by specifically blocking the β-cleavage of APP, but not the γ-cleavage of C99. Furthermore, in stark contrast to LME-tet, the best inhibitor among the previously identified ectodomain-targeting peptides (peptide #4) did not inhibit the conformational change of Aβ42 through β-sheet formation  than to Aβ40 (apparent Kd = 2.7 μM) in vitro and also efficiently binds Aβ42 released from cells. Of these two species, Aβ42 shows a greater propensity for aggregation, which leads to formation of the amyloid fibrils observed in senile plaquesion Fig. 53. Some AppNL-G-F/NL-G-F mice, suggesting that LME-tet may be efficacious for the treatment of AD. This is further supported by our finding that LME-tet binding to APP inhibits cleavage by β-secretase—the rate-limiting protease in Aβ production and a particularly attractive drug target15. Notably, a recent study reported that pharmacological inhibition of β-secretase is effective for limiting the initial process of plaque formation in vivo, but not for preventing subsequent plaque growth55. Thus, a key added benefit of LME-tet lies in its ability to inhibit, not only the formation of Aβ, but also its aggregation and polymerization, with high efficacy , mouse anti-human Aβ antibody 82E1 , rabbit anti-human Aβ42 specific antibody , mouse anti-Aβ17-24 antibody 4G8 , rabbit anti-sAPPβ antibody , rabbit anti-EEA1 antibody , rabbit polyclonal anti-LAMP1 antibody , rabbit polyclonal anti-SQSTM1/p62 antibody , rabbit polyclonal anti-LC3 antibody , rabbit polyclonal anti-β-actin antibody , rabbit anti-Sialyltransferase 1  antibody , mouse anti-Myc-Tag antibody 9B11 , horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG antibody , HRP-conjugated horse anti-mouse IgG antibody , Alexa Fluor 488-conjugated goat anti-mouse IgG antibody, , Alexa Fluor 546-conjugated goat anti-rabbit IgG antibody, , Alexa Fluor 546-conjugated goat anti-mouse IgG antibody .56. Fmoc-βAla-OH  was used in the first cycle, followed by aminohexanoic acid as a spacer. Fmoc-Lys(Fmoc)-OH  was used in the next two cycles to create four branches in the peptide chain for subsequent motif synthesis. Membranes were blocked with 5% skim milk and blotted with biotinylated Aβ1–28 (10 µg/ml) . After extensive washing, the Aβ1–28 bound to each tetravalent peptide was detected with horseradish peroxidase (HRP)-labeled streptavidin  and Enhanced Chemiluminescence (ECL) Plus Reagent . The intensity of each peptide spot was then quantified as a pixel value, using an ImageQuant LAS 500 CCD imager .Tetravalent peptide libraries were synthesized on a cellulose membrane, using a ResPep SL SPOT synthesizer N-α-Fmoc-protected amino acids and standard BOP/HOB coupling chemistry56. The terminal amino groups of tetravalent peptides were biotinylated with biotin  and 1-(bis[dimethylamino]methylene)-1H-benzotriazolium 3-oxide hexafluorophosphate  in the last cycle of peptide synthesis. The synthesized peptides were validated by mass spectrometry analysis, using the AutoflexII time-of-flight (TOF)/TOF system .Tetravalent and monomeric peptides were synthesized using 57 (7WD10-NotchΔE cells) were maintained in DMEM, supplemented with 10% FBS, 50 units/ml penicillin, 50 μg/ml streptomycin, 200 μg/ml G418 , and 250 μg/ml Hygromycin B . 7WD10-NotchΔE cells stably expressing β-galactoside α-2,6-sialyltransferase 1  were maintained in DMEM, supplemented with 10% FBS, 50 units/ml penicillin, 50 μg/ml streptomycin, 200 μg/ml G418, 250 μg/ml Hygromycin B , and 100 μg/ml Zeocin . All cell lines in this study were negative for mycoplasma contamination.Chinese Hamster Ovary (CHO) cells stably expressing human APP (APP1-751), referred to as 7WD10 cells, were maintained at 37 °C in Dulbecco’s Modified Eagle Medium , supplemented with 10% fetal bovine serum (FBS), 50 units/ml penicillin, 50 μg/ml streptomycin, and 200 μg/ml G418 . 7WD10 cells stably expressing Myc-tagged Notch with a deletion in the extracellular region (NotchΔE)7WD10, 7WD10-NotchΔE, or 7WD10-Notch ∆E-St6gal1 cells were seeded in 24-well plates and cultured at 37 °C, in the presence or absence of the indicated concentrations of tetravalent or monomeric peptides. After 48 h incubation, culture medium and cells were recovered separately. Cells were lysed in lysis buffer, containing 2% sodium dodecyl sulfate (SDS), 80 mM Tris-HCl (pH 6.8), 7% glycerol, 250 mM dithiothreitol (DTT), and 0.025% phenol red. Proteins in culture medium and lysates were separated by tricine SDS-polyacrylamide gel (12% acrylamide) electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes . To separate immature APP and mature APP, glycine SDS-PAGE (7% acrylamide) was used. After boiling in phosphate-buffered saline (PBS) for 5 min and then blocking with 5% skim milk, the membranes were immunoblotted with the indicated primary antibodies, followed by HRP-conjugated secondary antibodies. Protein bands were visualized using ECL Plus Reagent (PerkinElmer) and analyzed using an ImageQuant LAS 500 CCD imager . Total amounts of target proteins were measured using Image J software , after normalization to β-actin as an internal control.The indicated amounts of LME-tet (8.3 µM) or LME-mono (33 µM) were applied onto each well of a regular 96-well ELISA plate (Thermo Fisher Scientific) and incubated overnight for 24 h. After blocking, the plate was incubated with various concentrations of Aβ1–28, Aβ40, or Aβ42 (Peptide Institute) for 2 h at room temperature. Bound Aβ was detected using mouse monoclonal anti-human Aβ antibody 82E1  and HRP-conjugated horse anti-mouse IgG antibody .For coprecipitation from culture medium, 7WD10 cells in 12-well plates were cultured for 48 h at 37 °C, and the culture medium were recovered. This culture medium was treated with biotinylated LME-tet (50 µM) or LME-mono (200 µM) for 24 h at 4 °C, and then treated with streptavidin beads (Thermo Fisher Scientific) for 24 h at 4 °C. After extensive washing, the beads were analyzed by western blot, as described above. For coprecipitation from cell lysates, 7WD10 cells in 12-well plates were cultured for 30 min at 4 °C or 37 °C in the presence or absence of biotinylated LME-tet (50 µM) or LME-mono (200 µM). The cells were recovered and lysed in lysis buffer, as described above. Lysates were treated with streptavidin beads (Thermo Fisher Scientific) for 24 h at 4 °C, and after extensive washing, the beads were analyzed by western blot, as described above.Sub-confluent 7WD10 cells in 35-mm glass-bottom dishes were treated with mouse monoclonal anti-human APP antibody 6E10 (1:250 dilution) and biotinylated LME-tet (50 µM) on ice for 30 min. After washing, cells were incubated at 37 °C for the indicated times, fixed with 4% paraformaldehyde (PFA), and permeabilized with 0.2% TritonX-100. APP was detected using Alexa Fluor 546-conjugated goat anti-mouse IgG antibody. LME-tet was detected using Alexa Fluor 488-conjugated streptavidin. Early endosome antigen 1 (EEA1) was detected using rabbit polyclonal anti-EEA1 antibody, followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG antibody. Lysosome-associated membrane glycoprotein 1 (LAMP1) was detected using rabbit polyclonal anti-LAMP1 antibody, followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG antibody. For detection of the intracellular acidic compartment, cells were treated with 75 nM Lysotracker DND-99  for 1 h, followed by PFA fixation and permeabilization with 500 µg/ml digitonin . Fluorescent images were analyzed using LSM700 laser scanning confocal microscopy  and Image J software (NIH).7WD10 cells were treated with LME-tet (50 µM) or LME-mono (200 µM) in the presence or absence of lysosomal protease inhibitor cocktail containing 30 µM E-64 and 20 µM leupeptin for 48 h at 37 °C. The culture medium and cells were recovered separately, and proteins in each were analyzed by western blot, as described above.7WD10-NotchΔE-St6gal1 cells were treated with LME-tet or β-secretase inhibitor II   for 48 h. The culture medium was then analyzed by western blot, using anti-St6gal1 antibody to the detect β-cleavage product, St6gal1 fragment. 7WD10-NotchΔE cells were treated with LME-tet or γ-secretase inhibitor DAPT   for 48 h. The cell lysates were analyzed by western blot, using anti-Myc-Tag antibody to detect the γ-cleavage product, Notch intracellular domain (NICD).Escherichia coli BL21 cells and affinity-purified using anti-FLAG M2 beads (Sigma)45. Purified APP633–685-FLAG (50 nM) was incubated with β-secretase (Sigma) for 4 h, according to the manufacturer’s instructions, in the presence or absence of the indicated concentrations of LME-tet or LME-mono. β-cleaved C-terminal fragments (Ab33-FLAG) from APP633–685-FLAG were visualized by western blot with anti-human Aβ antibody 82E1 and quantified by Image J software (NIH).A fragment of human APP (position 633–685 in APP751), with N-terminal Myc and C-terminal FLAG tags (APP633–685-FLAG) was expressed in 58. FLAG-tagged C99 was prepared using the Bac-to-Bac Baculovirus Expression System  and used as a γ-secretase substrate47. FLAG-tagged C99 was incubated with enzyme in dilution buffer, containing 12.5 mM PIPES (pH 7.2), 62.5 mM sucrose, 0.25 mM ethylene glycol tetraacetic acid (EGTA), 0.25 mM diisopropyl fluorophosphate (DIFP), 2.5 µg/ml antipain, 2.5 µg/ml leupeptin, 2.5 µg/ml tosyl-L-lysyl-chloromethane hydrochloride (TLCK), 1.25 mM phenanthroline, 0.25 mM thiorphan, and 0.1% phosphatidylcholine at 37 °C for 4 h in the presence or absence the indicated concentrations of LME-tet. Production of Aβ in the reaction mixtures was measured by western blot analysis with anti-human Aβ antibody 82E1.γ-Secretase was prepared from the microsomal fraction of HEK293 cells, as previously described42 (20 μM) was incubated in the presence or absence of each peptide in 50 mM sodium phosphate buffer (pH 7.4) for the indicated time period at 37 °C. Far-UV CD spectra of Aβ42 were measured using a J-1500 spectrometer . Kinetic rates of β-sheet formation of Aβ42 were estimated by measuring time-dependent alterations of molar ellipticities of CD spectra at 217 nm60.Aβ42 (20 μM) was incubated in the presence or absence of each peptide in 50 mM sodium phosphate buffer (pH 7.4) for the indicated time period at 37 °C. Each Aβ solution (10 μL) was added to 800 μL of 20 μM Thioflavin T in 20 mM Tris-HCl (pH 8.0), and then subjected to excitation at 450 nm. Emission was monitored at 482 nm, using a FP-8300 fluorescence spectrometer (JASCO Inc.)59. Effects of LME-tet on growth of Aβ42 fibrils was measured as follows. To prepare the Aβ42 fibril-seed, Aβ42 (20 μM) was incubated in 50 mM sodium phosphate buffer (pH 7.4) for 2 days at 37 °C. Aβ42 (20 μM) was then incubated in the presence or absence of LME-tet (2 μM) in 50 mM sodium phosphate buffer (pH 7.4), with or without 2% (v/v) of the fibril-seed for the indicated time period at 37 °C. Each Aβ solution was analyzed as described above.Aβ42 (20 μM) was incubated in the presence or absence of each peptide in 50 mM sodium phosphate buffer (pH 7.4) at 37 °C for 3 days. Fibril samples (10 μL) were adsorbed on 400-mesh collodion-coated copper grids for 10 min and negatively stained with 2% phosphotungstic acid. Morphology of fibrils was analyzed using a transmission electron microscope (TEM), H-7650 , with at an accelerating voltage of 80 kV.AβApp KI, carrying Swedish (NL), Beyreuther/Iberian (F), and Arctic (G) mutations (AppNL-G-F/NL-G-F mice), were generated as described previously32. Mice were maintained under standard housing conditions, with lights on at 8:00 and lights off at 20:00, and animals were provided with water and food ad libitum. To increase stability of LME-tet in mice, acetylation of its terminal amino groups was performed using N-acetylimidazole, to generate Ac-LME-tet. Stabilized Ac-LME-tet in PBS (71 mg/kg) or PBS-only control was administered to 7-week-old mice intraperitoneally. After 7 days, mice were euthanized, and brains were removed. The cerebral cortex and olfactory bulb from one hemisphere were homogenized, extracted with a 4× volume of Tris-buffered saline solution (TBSs), containing 1% NP-40 and centrifuged at 100,000 × g for 1 h. The supernatant (1 ml) was subjected to immunoprecipitation with anti-human Aβ antibody 4G8, and then to western blot using anti-human Aβ antibody 82E1 . The indicated amounts of human Aβ1-40 (Peptide Institute) were used for the quantification. Data were analyzed using Mann–Whitney non-parametric statistics to assess significance. All animal experiments were approved by the Animal Ethics Committee of Doshisha University prior to their commencement and performed in accordance with approved protocols.Mice with single humanized U test was also used to analyze significant differences in data distribution between groups. In all cases, P < 0.05 was considered statistically significant. All statistical analysis was performed using IBM SPSS Statistics software (ver. 26.0.0.0). No statistical methods were used to determine the sample size. We repeated each experiment at least three times and confirmed the reproducibility of each result.Significant differences between each group and the control were analyzed using one-way analysis of variance (ANOVA), followed by Dunnett’s test. The non-parametric Mann–Whitney Further information on research design is available in the Peer Review FileSupplementary FiguresDescription of Additional Supplementary FilesSupplementary Data 1Reporting Summary


Stimulation of CXCR4+ / CXCR7+-GCT cells with CXCL12 resulted in an enhanced proliferative and migratory capacity, while this effect could be reverted using CXCR4 inhibitors or a CXCR7-nanobody. Molecularly, the CXCR4 / CXCR7-signaling cascade could be activated independently of MAPK (ERK1 / 2)-phosphorylation. Although, in CXCR4− / CXCR7−-embryonal carcinoma cells, CXCR7-expression was re-induced upon inhibition of ERK1 / 2-signaling. This study identified a nanobody-drug-conjugate targeting CXCR4 as a putative therapeutic option for GCT, i.e. seminoma and yolk-sac tumors. Furthermore, this study shed light on the functional role of the CXCR4 / CXCR7 / CXCL12-signaling cascade in GCT, demonstrating an important influence on proliferation and migration.Being stimulated by the chemokine CXCL12, the CXCR4 / CXCR7 cascade is involved in tumor proliferation, migration, and metastasis. The interaction between CXCL12, secreted by cells from the microenvironment, and its receptors is complex and has been ascribed to promote chemotherapy resistance. However, the role of this signaling axis and its targetability in germ cell tumors (GCT) is not fully understood. Thus, this study investigated the therapeutic efficacy of a nanobody-drug-conjugate targeting CXCR4 (CXCR4-NDC) and functionally characterized this signaling pathway in GCT using small molecule inhibitors and nanobodies. As shown by diminished cell viability, enhanced apoptosis induction, and detection of mitotic catastrophes, we confirmed the cytotoxic efficacy of the CXCR4-NDC in CXCR4The online version contains supplementary material available at 10.1186/s40164-023-00460-9. GCT account for 1–2% of all neoplasms occurring in the male population, but represent the most common cancer type in young men [CXCR4 / CXCR7 were not found in GCT  as CXCR4− / CXCR7−, choriocarcinoma (CC) as CXCR4− / CXCR7+, and yolk-sac tumors (YST) as CXCR4+ / CXCR7+ , similar observations were found on mRNA level  , mutations in GCT Fig. A. On proR7+ Fig. A. In mix7+) Fig. B. CXCR7 L12 Fig. C.50 concentrations for the CXCR4-NDC were between 56.4 and 310.6 nM for GCT cells (including cisplatin-resistant sub clones (-R)) and > 500 nM for fibroblasts, thereby opening a therapeutic window  . Treatmells Fig. D. In fiblls Fig. D.FOS and MMP3 in GCT72CXCR4+/CXCR7+ and BeWoCXCR7+ cells, while BeWo cells further showed increased expression of CD44, IL6, ITGA4A, and MMP1   of β-Catenin and STAT5a / b (Y699), in 1411HCXCR4+/CXCR7+ elevated p-GSK-3α / β (S21 / S9), p-p53 (S392) and p-WNK1 (T60), and in BeWoCXCR7+ enhanced levels of p-GSK-3α / β (S21 / S9), p-p53 (S392 / S46), p-SRC (Y419) and p-WNK1 (T60). In GCT72CXCR4+/CXCR7+ cells, phosphorylation of thirteen signaling-molecules, including ERK1 / 2 , GSK-3α / β (S21 / S9), β-Catenin, and p53 (S15 / S46) was reduced, while p-p53 (S392) was strongly enhanced. Phosphorylation of p53 at S15 has been described to increase stability of p53 by blocking MDM2 binding [p53AIP1 [Next, we deciphered the molecular role of this signaling axis on mRNA and phospho-proteome level. Treatment with recombinant CXCL12  resulted in enhanced levels of MP1 Fig. E. Furtheml) Fig. F. In TCa CXCL12 20 ng / ml CXCL12 20 ng / ml and p53 5 / S46 wCXCR4+/CXCR7+ and 1411HCXCR4+/CXCR7+ cells was enhanced upon CXCL12-stimulation; this effect could be blocked upon application of a CXCR7-blocking nanobody (CXCR7-NB) or CXCR4-inhibitors  , while increasing CXCR7 expression (and CXCR4 in NT2/D1)  Fig. F. Thus, + SEM and YST  Expression profile (RPKM) of CXCR4 / CXCR7 in GCT cells based on previously published RNA sequencing data . C) CXCL12-ELISA of supernatants from fibroblasts (MPAF) and GCT cells NCCIT and JAR. D) Flow cytometry data indicating changes in the cell cycle distribution of GCT cell lines and MPAF treated with the CXCR4-NBC (blue) in comparison to CXCR4-NB controls (grey). E) Expression of BCL2, CD44, COL3A1, CCND1, EGFR, FOS, IL6, ITGA4, MMP1/2/3, SRC, TGFB1/2/3, VEGFA in CXCL12-stimulated  GCT72, 1411H, TCam-2, and BeWo cells as compared to their respective untreated controls. F) Raw data of a human phospho-kinase array of various cell lysates  treated with recombinant CXCL12 (250 ng / ml) for 24 h. Untreated cells served as controls. G) Western blot analyses of pERK and total ERK of EC cells  treated daily with 100 nM ERK inhibitor SCH772984 for 96 hSupplementary Material 1: Fig. S1: A) Mutational status and mRNA expression profile of Supplementary Material 2: Supplemental ‘Material & Methods’Supplementary Material 3: Table S1: A) cell lines, B) drugs, C) antibodies, and D) oligonucleotides used in this study


Foi relatado que o RNA 1 antisenso 1 (SLC26A4-AS1) do membro 4 da família de transportadores de soluto 26 está altamente relacionado à hipertrofia cardíaca. Esta pesquisa visa investigar o papel e o mecanismo específicos de SLC26A4-AS1 na hipertrofia cardíaca, fornecendo um novo marcador para o tratamento da hipertrofia cardíaca. Angiotensina II (AngII) foi infundida em cardiomiócitos ventriculares (NMVCs) de camundongos neonatos para induzir hipertrofia cardíaca. A expressão gênica foi detectada por PCR quantitativo em tempo real (RT-qPCR). Os níveis de proteína foram avaliados por western blot. Ensaios funcionais analisaram o papel de SLC26A4-AS1. O mecanismo de SLC26A4-AS1 foi avaliado por imunoprecipitação de proteína de ligação a RNA (RIP), pull-down de RNA e ensaios de luciferase repórter. O valor de p < 0,05 foi identificado como significância estatística. O teste t de Student avaliou a comparação dos dois grupos. A diferença entre os diferentes grupos foi analisada por análise de variância (ANOVA) de uma via. SLC26A4-AS1 é regulado para cima em NMVCs tratados com AngII e promove hipertrofia cardíaca induzida por AngII. SLC26A4-AS1 regula o membro 4 da família de transportadores de soluto 26 (SLC26A4) por meio do funcionamento como um RNA endógeno competitivo (ceRNA) para modular o microRNA (miR)-301a-3p e o miR-301b-3p em NMVCs. SLC26A4-AS1 promove hipertrofia cardíaca induzida por AngII via regulação para cima de SLC26A4 ou absorção de miR-301a-3p/miR-301b-3p. SLC26A4-AS1 agrava a hipertrofia cardíaca induzida por AngII via absorção de miR-301a-3p ou miR-301b-3p para aumentar a expressão de SLC26A4. Apesar disso, a hipertrofia miocárdica contínua é uma doença cardíaca típica induzida pelo aumento da carga de trabalho do coração acompanhada de remodelação cardíaca mal adaptativa, levando à estenose aórtica e cardiomiopatia dilatada. Paredes ventriculares excessivamente espessas e cardiomiócitos são os padrões primários de hipertrofia cardíaca. Nos últimos anos, a hipertrofia cardíaca tornou-se um problema urgente que pode levar a uma incidência crescente de insuficiência cardíaca e morte súbita.A hipertrofia cardíaca é uma resposta adaptativa para melhorar a pós-carga cardíaca para sustentar a função cardíaca sistólica nos estágios iniciais. Estudos recentes provaram que muitos fatores, incluindo fatores de crescimento específicos, hormônios peptídicos e microRNAs (miRNAs), são responsáveis pela hipertrofia cardíaca, mas o mecanismo molecular exato subjacente à progressão da hipertrofia cardíaca permanece obscuro.As características marcantes da hipertrofia cardíaca patológica são a elevação de genes fetais como o peptídeo natriurético cerebral (BNP), peptídeo natriurético atrial (ANP) e a cadeia pesada de beta-miosina (β-MHC). Evidências crescentes revelaram os papéis vitais dos lncRNAs no desenvolvimento de doenças cardiovasculares, incluindo a hipertrofia cardíaca. Por exemplo, o lncRNA SNHG14 induzido por SP1 promove hipertrofia cardíaca via rede miR-322-5p/miR-384-5p/PCDH17 em cardiomiócitos induzidos por angiotensina II (AngII). O silenciamento de LncRNA MIAT restringe a hipertrofia cardíaca induzida por AngII via eixo de RNA endógeno concorrente miR-93/TLR4 (ceRNA). O membro 4 da família LncRNA transportadora de soluto 26 RNA antisenso 1 (SLC26A4-AS1) foi relatada como altamente associada com hipertrofia cardíaca.Os RNAs longos não codificantes (lncRNAs) participam de processos patológicos por meio do controle da expressão gênica nos níveis epigenético, transcricional e pós-transcricional. Os miRNAs geralmente exercem funções celulares por meio do direcionamento de RNAs mensageiros a jusante (mRNAs). Numerosos miRNAs têm sido apontados como reguladores essenciais na progressão da hipertrofia cardíaca. Por exemplo, o miR-17-5p facilita a hipertrofia cardíaca induzida por AngII por meio da inibição da expressão de Mfn2 e ativação do eixo PI3K/AKT/mTOR para suprimir a autofagia. O MiR-29a melhora a hipertrofia cardíaca induzida pelo cloridrato de isoproterenol ao reprimir a expressão do PPARδ. Considerando o papel vital dos miRNAs na hipertrofia cardíaca, tentamos investigar o mecanismo regulador do SLC26A4-AS1 focando nos miRNAs que interagem com ele.Os miRNAs são outro tipo de RNA não codificante com um comprimento de cerca de 22 nucleotídeos.No estudo atual, o efeito de SLC26A4-AS1 no nível de BNP, ANP e β-MHC foi explorado para determinar a função de SLC26A4-AS1 na hipertrofia cardíaca induzida por AngII. O mecanismo regulador de SLC26A4-AS1 foi investigado através do eixo ceRNA. Nosso estudo pode oferecer uma nova visão sobre os mecanismos fundamentais do lncRNA na hipertrofia cardíaca, fornecendo um novo alvo para o tratamento da hipertrofia cardíaca.Os cardiomiócitos foram isolados dos camundongos recém-nascidos. O comitê de ética do nosso Hospital aprovou experimentos com animais. Para induzir a hipertrofia dos cardiomiócitos, os corações extraídos foram partidos em pedaços e tratados com tripsina 0,25% , seguido de incubação em meio Eagle modificado por Dulbecco/F-12  com 10% FBS . Após a confluência celular atingir 80%, as células nas placas foram transferidas para placas de cultura e inoculadas com AngII  com a concentração de 150 nM para 1, 6, 12 e 24 h para estimular fenótipos hipertróficos de cardiomiócitos, estabelecendo o modelo celular de hipertrofia cardíaca.Foram todos adquiridos da GenePharma Co., Ltd.  grampos curtos de RNA (shRNAs) contra SLC26A4-AS1 , shRNAs contra o membro 4 da família 26 de transportador de soluto (SLC26A4) , controle negativo de shRNA , pcDNA3.1-SLC26A4 , vetor vazio pcDNA3.1 , microRNA (miR)-301a-3p mimético , miméticos de miR-301b-3p , controle negativo de miméticos de miRNA , inibidor de miR-301a-3p , inibidor de miR-301b-3p  e controle negativo do inibidor de miRNA . Todas as transfecções celulares foram aplicadas usando Lipofectamine 3000 , seguido pelo tratamento de 150 nM AngII por 24 h. Três ensaios independentes foram conduzidos. A sequência do iniciador para SLC26A4-AS1 e GAPDH é mostrada naO RNA total das células foi extraído pelo reagente TRIzol . A concentração total de RNA foi determinada medindo o valor de absorção a 260 nm com um microespectrofotômetro. O RNA (1μl) foi transcrito reversamente para compor o DNA complementar usando a transcriptase reversa M-MLV . Em seguida, a qPCR foi realizada com o kit SYBR Premix Ex Taq™ II . A expressão de miRNA foi normalizada para U6, e a de lncRNA e mRNA foi normalizada para gliceraldeído-3-fosfato desidrogenase (GAPDH), que foi baseado no método 2−ΔΔCt. A análise por RT-qPCR foi realizada conforme descrito anteriormente.Foi usado tampão de lise RIPA  para extrair proteínas totais, e a concentração de proteína foi verificada usando um kit de ensaio de proteína BCA . Após o tratamento de eletroforese em gel de poliacrilamida e dodecil sulfato de sódio , as proteínas foram transferidas para membrana de PVDF  e bloqueadas em leite desengordurado a 5%. Posteriormente, a membrana foi cultivada com anticorpos primários contra ANP , BNP , β-MHC , SLC26A4  e GAPDH  a 4°C durante a noite. Após a lavagem, a membrana foi cultivada com anticorpo secundário conjugado com HRP  por 1 h. O sinal foi medido usando o substrato de western blotting de quimioluminescência melhorada. Todos os experimentos independentes foram repetidos três vezes.As células foram lavadas e fixadas e bloqueadas com soro de cabra normal contendo 1% de BSA . Posteriormente, as células foram cultivadas com anti-α-actinina  a 4°C por uma noite inteira e foram incubadas com um anticorpo secundário por 1 h. DAPI  foi aplicado para corar os núcleos, e um microscópio de fluorescência foi utilizado para capturar as imagens e avaliar a área de superfície celular. Três ensaios independentes foram conduzidos.Um kit PARIS  foi usado para isolar as frações citoplasmática e nuclear de cardiomiócitos ventriculares (NMVCs) de camundongos neonatos. A expressão de GAPDH, U6 ou SLC26A4-AS1 no núcleo e no citoplasma foi avaliada por análise de RT-qPCR. Três ensaios independentes foram conduzidos.As sondas SLC26A4-AS1 marcadas com Alexa Fluor 555  foram sintetizadas pela RiboBio . Os experimentos FISH foram implementados com um kit FISH (RiboBio). As células foram semeadas em lâminas por 24 horas de incubação. Após fixação e permeabilização, as células foram cultivadas com sondas SLC26A4-AS1 a 37°C durante a noite. O DAPI foi utilizado para corar os núcleos das células e as imagens foram observadas em microscópio de fluorescência. Bio-repetições foram realizadas em triplicado.Com base nas instruções, o kit Magna RIP de imunoprecipitação de proteína de ligação a RNA (Millipore) e o anticorpo Ago2  foram usados para realizar ensaios RIP. Os RNAs coprecipitados foram analisados via RT-qPCR. Três ensaios independentes foram conduzidos.Os NMVCs foram transfectados com uma sonda SLC26A4-AS1 biotinilada por 48 h. Em seguida, as células coletadas foram cultivadas com esferas magnéticas de estreptavidina M-280 . Os RNAs ligados foram avaliados via RT-qPCR. Três ensaios independentes foram conduzidos.4/poço) foram cultivadas em 24 placas poço para incubação. As células foram então transfectadas com esses plasmídeos repórteres e miméticos de miR-301a-3p ou miméticos de miR-301b-3p, respectivamente. Após 48 h de transfecção usando o reagente Lipofectamine 3000, um kit Dual-Luciferase Reporter Assay (Promega) foi aplicado para medir a atividade relativa da luciferase. Três ensaios independentes foram conduzidos.A sequência de SLC26A4-AS1 ou SLC26A4 3’UTR contendo o local de ligação previsto com miR-301a-3p e miR-301b-3p foi clonada várias vezes no vetor de luciferase dupla pmirGLO , bem como o vetor putativo e sequências mutantes. As células , onde δ é a diferença mínima, σ é o desvio padrão total, α é o nível de inspeção , e β é a taxa de erro tipo II . Os dados foram apresentados como a média ± desvio padrão para 3 ensaios independentes. O software GraphPad Prism versão 5.0 foi aplicado para analisar os dados. A normalidade dos dados foi verificada por meio do teste de Shapiro-Wilk, que mostrou que todos os dados seguiram a distribuição normal. A significância estatística entre os grupos foi investigada pelo teste t de Student não pareado ou análise de variância unidirecional, seguido de um teste post hoc (Tukey e Dunnett). Além disso, um valor de p menor que 0,05 foi identificado como estatisticamente significativo.Para avaliar o papel específico de SLC26A4-AS1 na hipertrofia cardíaca, primeiro tratamos NMVCs com AngII para induzir fenótipos hipertróficos de cardiomiócitos. Os níveis de biomarcadores hipertróficos  em NMVCs tratados com AngII aumentaram gradualmente ao longo do tempo . A coloAlém disso, a análise de RT-qPCR verificou a transfecção bem-sucedida de plasmídeos sh-SLC26A4-AS1#1/2 em NMVCs induzidos por AngII . A área Conforme mostrado naFigura Suplementar S3A, o banco de dados UCSC (http://genome.ucsc.edu/) previu que SLC26A4 era um gene próximo de SLC26A4-AS1. Portanto, conjecturamos que SLC26A4-AS1 pode regular SLC26A4 para afetar a hipertrofia cardíaca induzida por AngII. Em seguida, ensaios Ago2-RIP foram realizados para que Ago2 seja o componente central do complexo de silenciamento induzido por RNA. SLC26A4-AS1 e SLC26A4 foram altamente abundantes em precipitados ligados a anti-Ago2 . A regulação para baixo de SLC26A4 reverteu o aumento da área de superfície de NMVCs tratados com AngII  e a regulação para cima dos níveis de biomarcadores hipertróficos estimulados pelo tratamento com AngII . Coletivamente, SLC26A4 promove hipertrofia cardíaca induzida por AngII in vitro.Para detectar a função de SLC26A4 na hipertrofia cardíaca induzida por AngII, foram realizados ensaios de perda de função após a verificação da eficiência de sh-SLC26A4#1/2 em NMVCs tratados com AngII , descobrimos que a área de superfície celular e os níveis de biomarcadores hipertróficos em NMVCs induzidos por AngII foram acentuadamente diminuídos por miR-301a -3p ou superexpressão de miR-301b-3p . Todos esses dados sugeriram que miR-301a-3p ou miR-301b-3p poderiam suprimir a hipertrofia cardíaca induzida por AngII in vitro.É bem reconhecido que os lncRNAs podem funcionar como concorrentes de RNAs endógenos (ceRNAs) para regular a expressão gênica por meio da interação com miRNAs em níveis pós-transcricionais.SLC26A4 . MiR-30SLC26A4 . Além dSLC26A4 , indicaSLC26A4 . Após aFigura Suplementar S3B, os respectivos locais de ligação dos dois miRNAs em SLC26A4-AS1 foram previstos a partir do banco de dados starBase. MiR-301a-3p ou miR-301b-3p superexpressos reduziram significativamente a atividade de SLC26A4-AS1 de tipo selvagem em vez de SLC26A4-AS1-MUT , are accountable for cardiac hypertrophy,but the exact molecular mechanism underlying the progression of cardiac hypertrophy remains unclear.The outstanding characteristics of pathologic cardiac hypertrophy are the elevation of fetal genes such as brain natriuretic peptide (BNP), atrial natriuretic peptide (ANP), and beta-myosin heavy chain (β-MHC).Growing evidence has unveiled the vital roles of lncRNAs in cardiovascular disease development, including cardiac hypertrophy.For instance, SP1-induced lncRNA SNHG14 promotes cardiac hypertrophy via miR-322-5p/miR-384-5p/PCDH17 network in Angiotensin II (AngII)-induced cardiomyocytes.LncRNA MIAT silencing restrains AngII-induced cardiac hypertrophy via miR-93/TLR4 competing endogenous RNA (ceRNA) axis.LncRNA solute carrier family 26 member 4 antisense RNA 1 (SLC26A4-AS1) has been reported as highly associated with cardiac hypertrophy.Long non-coding RNAs (lncRNAs) participate in pathological processes via governing gene expression at epigenetic, transcriptional, and post-transcriptional levels.MiRNAs usually exert cellular functions via targeting downstream messenger RNAs (mRNAs).Numerous miRNAs have been pointed out as essential regulators in the progression of cardiac hypertrophy.For example, miR-17-5p facilitates AngII-induced cardiac hypertrophy via inhibiting Mfn2 expression and activating the PI3K/AKT/mTOR axis to suppress autophagy.MiR-29a relieves isoproterenol hydrochloride-induced cardiac hypertrophy by repressing PPARδ expression.Considering the vital role of miRNAs in cardiac hypertrophy, we tried to investigate the regulatory mechanism of SLC26A4-AS1 by focusing on the miRNAs that interact with it.MiRNAs are another type of non-coding RNA with a length of about 22 nucleotides.In the current study, the effect of SLC26A4-AS1 on the level of BNP, ANP, and β-MHC was explored to determine the function of SLC26A4-AS1 in AngII-induced cardiac hypertrophy. The regulatory mechanism of SLC26A4-AS1 was investigated via the ceRNA axis. Our study might offer a novel insight into the fundamental mechanisms of lncRNA in cardiac hypertrophy, providing a novel target for cardiac hypertrophy treatment.Cardiomyocytes were isolated from the neonatal mice. The ethics committee of our Hospital approved animal experiments. To induce hypertrophy of cardiomyocytes, the extracted hearts were split into pieces and treated with 0.25% trypsin , followed by incubation in Dulbecco’s modified Eagle medium/F-12  with 10% FBS . After cell confluence reached 80%, cells in the dishes were transferred to culture plates and inoculated with AngII  with the concentration of 150 nM for 1, 6, 12, and 24 h to stimulate hypertrophic phenotypes of cardiomyocytes, establishing the cell model of cardiac hypertrophy.Short hairpin RNAs (shRNAs) against SLC26A4-AS1 , shRNAs against solute carrier family 26 member 4 (SLC26A4) , negative control of shRNA , pcDNA3.1-SLC26A4 , pcDNA3.1 empty vector , microRNA (miR)-301a-3p mimics , miR-301b-3p mimics , , miR-301a-3p inhibitor , miR-301b-3p inhibitor  and negative control of miRNA inhibitor  were all purchased from GenePharma Co., Ltd. . All cell transfections were applied using Lipofectamine 3000 , followed by the treatment of 150 nM AngII for 24 h.Ctmethod. RT-qPCR analysis was performed as described previously.Three independent assays were conducted. The primer sequence for SLC26A4-AS1 and GAPDH is shown inTotal RNA from cells was extracted by TRIzol  reagent. The total RNA concentration was determined by measuring the absorption value at 260nm with a microspectrophotometer. The RNA (1μl) was reversely transcribed to compose complementary DNA using M-MLV reverse transcriptase . Afterward, qPCR was conducted with the SYBR Premix Ex Taq™ II kit . The expression of miRNA was normalized to U6, and that of lncRNA and mRNA was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which was based on the 2RIPA lysis buffer  was used to extract total proteins, and protein concentration was verified using a BCA protein assay kit . After the treatment of sodium dodecyl sulfate-polyacrylamide gel electrophoresis , proteins were transferred onto PVDF membrane  and blocked in 5% defatted milk. Subsequently, the membrane was cultured with primary antibodies against ANP , BNP , β-MHC , SLC26A4  and GAPDH  at 4°C overnight. After being washed, the membrane was cultured with HRP-conjugated secondary antibody  for 1 h. The signal was measured using the enhanced chemiluminescence western blotting substrate. All independent experiments were repeated three times.Cells were washed and fixed and were blocked with normal goat serum containing 1% BSA . Subsequently, cells were cultured with anti-α-actinin  at 4°C for a whole night and were further hatched with a secondary antibody for 1 h. DAPI  was applied to stain the nuclei, and a fluorescence microscope was used to capture the images and assess the cell surface area. Three independent assays were conducted.A PARIS kit  was applied for isolating the cytoplasmic and nuclear fractions from neonatal mouse ventricular cardiomyocytes (NMVCs). GAPDH, U6, or SLC26A4-AS1 expression in the nucleus and cytoplasm was assessed by RT-qPCR analysis. Three independent assays were conducted.Alexa Fluor 555-labeled SLC26A4-AS1 probes  were synthesized by RiboBio . FISH experiments were implemented with a FISH kit (RiboBio). Cells were seeded into slides for 24 h of incubation. After fixation and permeabilization, cells were cultured with SLC26A4-AS1 probes at 37°C overnight. DAPI was utilized to stain cell nuclei, and the images were observed under a fluorescence microscope. Bio-repeats were conducted in triplicate.Based on the instructions, the Magna RIP RNA-binding protein immunoprecipitation kit (Millipore) and the Ago2 antibody  were used to perform RIP assays. Co-precipitated RNAs were analyzed via RT-qPCR. Three independent assays were conducted.NMVCs were transfected with a biotinylated SLC26A4-AS1 probe for 48 h. Then collected cells were cultured with M-280 streptavidin magnetic beads . The bound RNAs were assessed via RT-qPCR. Three independent assays were conducted.4/well) were grown in 24-well plates for incubation. Cells were then transfected with these reporter plasmids and miR-301a-3p mimics or miR-301b-3p mimics, respectively. After 48 h transfection using Lipofectamine 3000 reagent, a Dual-Luciferase Reporter Assay kit (Promega) was applied to measure the relative luciferase activity. Three independent assays were conducted.The sequence of SLC26A4-AS1 or SLC26A4 3’UTR containing the predicted binding site with miR-301a-3p and miR-301b-3p were severally cloned into the pmirGLO dual luciferase vector , as well as the putative and mutated sequences. Cells , where δ is the minimum difference, σ is the total standard deviation, α is inspection level (=0.05), and β is the rate of type II mistake (=0.10). Data were shown as the mean ± standard deviation for 3 independent assays. GraphPad Prism Version 5.0 Software was applied to analyze data. Data normality was checked using the Shapiro-Wilk test, which showed that all data followed the normal distribution. The statistical significance among groups was investigated by the unpaired Student’s t-test or one-way analysis of variance, followed by a post hoc test (Tukey and Dunnett). Besides, a p value less than 0.05 was identified as statistically significant.To evaluate the specific role of SLC26A4-AS1 in cardiac hypertrophy, we first treated NMVCs with AngII to induce hypertrophic phenotypes of cardiomyocytes. The levels of hypertrophic biomarkers  in AngII-treated NMVCs were gradually increased over time . StainiFurthermore, RT-qPCR analysis verified the successful transfection of sh-SLC26A4-AS1#1/2 plasmids in AngII-induced NMVCs . The inAs shown inFigure Supplementary S3A, the UCSC database (http://genome.ucsc.edu/) predicted that SLC26A4 was a nearby gene of SLC26A4-AS1. Therefore, we conjectured that SLC26A4-AS1 might regulate SLC26A4 to affect AngII-induced cardiac hypertrophy. Then, Ago2-RIP assays were performed for that Ago2 is the central component of RNA-induced silencing complex. SLC26A4-AS1 and SLC26A4 were highly abundant in anti-Ago2 bound precipitates . SLC26A4 downregulation reversed the increased surface area of AngII-treated NMVCs  and the upregulation of hypertrophic biomarker levels stimulated by AngII treatment . Collectively, SLC26A4 promotes AngII-induced cardiac hypertrophy in vitro.To detect the function of SLC26A4 in AngII-induced cardiac hypertrophy, loss-of-function assays were performed after the efficiency of sh-SLC26A4#1/2 in AngII-treated NMVCs was verified , we found that the cell surface area and hypertrophic biomarker levels in AngII-induced NMVCs were markedly decreased by miR-301a-3p or miR-301b-3p overexpression . All these data suggested that miR-301a-3p or miR-301b-3p could suppress AngII-induced cardiac hypertrophy in vitro.It is well-acknowledged that lncRNAs can function as competing for endogenous RNAs (ceRNAs) to regulate gene expression via interacting with miRNAs at post-transcriptional levels.SLC26A4 . MiR-30SLC26A4 . FurtheSLC26A4 , indicaSLC26A4 . After Figure Supplementary S3B, the respective binding sites of the two miRNAs on SLC26A4-AS1 were predicted from the starBase database. Overexpressed miR-301a-3p or miR-301b-3p significantly reduced the activity of wild-type SLC26A4-AS1 rather than that of SLC26A4-AS1-MUT (Figure Supplementary S3B. The luciferase activity of SLC26A4 3’UTR wild-type was decreased in AngII-induced NMVCs after the overexpression of miR-301a-3p or miR-301b-3p (As shown inAS1-MUT . The bi301b-3p . Additi301b-3p . To sumTo explore whether SLC26A4-AS1 affects AngII-induced cardiac hypertrophy via regulating miR-301a-3p/miR-301b-3p, we firstly inhibited the expression of miR-301a-3p and miR-301b-3p in AngII-induced NMVCs . The deIn recent years, many researchers have revealed that lncRNAs are important regulators in the progression of pathological cardiac hypertrophy. LncRNA PEG10 is upregulated in phenylephrine-treated primary cardiomyocytes and exacerbates phenylephrine-induced cardiac hypertrophy through modulating HOXA9.LncRNA TINCR relieves AngII-induced cardiac hypertrophy via down-regulating CaMKII.LncRNA TUG1 is upregulated in AngII-treated hypertrophic H9c2 cells and promotes AngII-induced cardiac hypertrophy via interacting with miR-29b-3p.In the present study, we found that a novel lncRNA SLC26A4-AS1 is highly associated with cardiac hypertrophy10and upregulated in AngII-induced hypertrophic cardiomyocytes. Furthermore, SLC26A4-AS1 silencing impaired cell surface area and the level of ANP, BNP, and β-MHC in AngII-induced hypertrophic cardiomyocytes. SLC26A4-AS1 has been associated with the development of several cancers, such as papillary thyroid carcinomaand glioma,but its role and potential mechanism in cardiac hypertrophy remain largely unclear. In this research, we verified the promotion of SLC26A4-AS1 on AngII-induced cardiac hypertrophy in vitro for the first time.Cardiac hypertrophy is divided into physiological and pathological hypertrophy. Pathological cardiac hypertrophy is the main induction factor for the progression of heart failure with abnormal expressions of cardiac genes, altered cardiac morphology, and maladaptive cardiac remodeling.Consistent with this evidence, this research confirmed that silencing SLC26A4 suppresses AngII-induced cardiac hypertrophy. Moreover, SLC26A4 overexpression could offset the suppressive effect of SLC26A4-AS1 depletion on AngII-induced cardiac hypertrophy.Another finding of this research was that SLC26A4-AS1 regulates its nearby gene SLC26A4 to affect AngII-induced cardiac hypertrophy. SLC26A4 has been reported to increase cardiac hypertrophy.Li et al. have demonstrated that lncRNA MIAT accelerates AngII-induced cardiac hypertrophy through modulating miR-93 and TLR4.Wo et al. have uncovered that CHRF promotes isoproterenol-induced cardiac hypertrophy by targeting miR-93 and Akt3.The current study also demonstrated that SLC26A4-AS1 regulates SLC26A4 expression via functioning as ceRNAs to sponge miRNAs at a post-transcriptional level. After finding the common miRNAs of SLC26A4-AS1 and SLC26A4, we revealed that SLC26A4-AS1 regulates SLC26A4 via functioning as ceRNAs to sponge miR-301a-3p or miR-301b-3p.Recent studies have demonstrated that lncRNAs could work by ceRNA network to drive cardiac hypertrophy, in which lncRNA serves as a miRNA sponge to regulate the expression of mRNAs. Zhou et al. have proved that lncRNA UCA1 facilitates cardiac hypertrophy progression by competitively modulating miR-184 to affect HOXA9 expression.MiR-625-5p represses cardiac hypertrophy via interacting with CaMKII and STAT3.MiR-29a facilitates cardiac hypertrophy via the PTEN/AKT/mTOR pathway. MiR-301a-3p and miR-301b-3p have also been reported to be implicated in human cancers, such as hepatocellular carcinoma,non-small cell lung cancer,and pancreatic cancer.Nonetheless, the suppressive role of miR-301a-3p and miR-301b-3p in AngII-induced cardiac hypertrophy was proved for the first time. Additionally, inhibition of miR-301a-3p or miR-301b-3p could reverse the repressive effects of SLC26A4-AS1 silencing on AngII-induced cardiac hypertrophy.Previous studies have uncovered that miRNAs exert vital functions in cardiac hypertrophy. MiR-22 acts as a vital modulator of cardiomyocyte hypertrophy and cardiac remodeling.In conclusion, this research found that SLC26A4-AS1 regulates the miR-301a-3p/miR-301b-3p/SLC26A4 axis to facilitate cardiac hypertrophy induced by AngII, which shed light on a promising biomarker for cardiac hypertrophy treatment.Nonetheless, the current study was performed only in vitro experiments to investigate the effect of SLC26A4-AS1 in cardiomyocytes, which poses certain limitations. To improve our understanding of the specific role and underlying mechanism of SLC26A4-AS1 in cardiac hypertrophy, we will perform in vivo experiments and clinicopathological analysis of SLC26A4-AS1 in cardiac hypertrophy in our future research.


Flavobacterium johnsoniae and hypothesized that FjDexUL is involved in the degradation of α-(1→2)-branched dextran. In this study, we demonstrate that FjDexUL proteins recognize and degrade α-(1→2)- and α-(1→3)-branched dextrans produced by Leuconostoc citreum S-32 (S-32 α-glucan). The FjDexUL genes were significantly upregulated when S-32 α-glucan was the carbon source compared with α-glucooligosaccharides and α-glucans, such as linear dextran and branched α-glucan from L. citreum S-64. FjDexUL glycoside hydrolases synergistically degraded S-32 α-glucan. The crystal structure of FjGH66 shows that some sugar-binding subsites can accommodate α-(1→2)- and α-(1→3)-branches. The structure of FjGH65A in complex with isomaltose supports that FjGH65A acts on α-(1→2)-glucosyl isomaltooligosaccharides. Furthermore, two cell surface sugar-binding proteins (FjDusD and FjDusE) were characterized, and FjDusD showed an affinity for isomaltooligosaccharides and FjDusE for dextran, including linear and branched dextrans. Collectively, FjDexUL proteins are suggested to be involved in the degradation of α-(1→2)- and α-(1→3)-branched dextrans. Our results will be helpful in understanding the bacterial nutrient requirements and symbiotic relationships between bacteria at the molecular level.Dextran is an α-(1→6)-glucan that is synthesized by some lactic acid bacteria, and branched dextran with α-(1→2)-, α-(1→3)-, and α-(1→4)-linkages are often produced. Although many dextranases are known to act on the α-(1→6)-linkage of dextran, few studies have functionally analyzed the proteins involved in degrading branched dextran. The mechanism by which bacteria utilize branched dextran is unknown. Earlier, we identified dextranase (FjDex31A) and kojibiose hydrolase (FjGH65A) in the dextran utilization locus (FjDexUL) of a soil Bacteroidota  Bacteroides thetaiotaomicron, a human intestinal bacterium   and polysaccharide lyases, to degrade and utilize carbohydrates depending on their habitat. Bacteria belonging to the Bacteroidota phylum possess polysaccharide utilization loci (PUL) that are involved in the systematic recognition and degradation of glycans and transport of the degradation products. PULs encode proteins such as cell surface sugar-binding proteins, sugar transporters, carbohydrate-active enzymes, such as GH and polysaccharide lyases, and transcriptional regulators . The stuacterium , 3, 4. Sacterium , 6, 7. Tacterium , 10, 11./PULDB/) . In receen found , 19, 20.en found .Leuconostoc and Streptococcus spp. vary depending on the species and strains, and branched dextran with α-(1→2)- and α-(1→3)-linkages have been reported (Leuconostoc mesenteroides NRRL B-512 is composed of 95% α-(1→6)-linkages and 5% α-(1→3)-linkages -linkages in the main chain, with a relatively large proportion of α-(1→2)-branches (23%–36%) and a small number of α-(1→3)-linkages - and α-(1→3)-linkages -linkages in addition to α-(1→6)-linkages; moreover, enzyme digestion analysis showed that it contains α-(1→2)-linked glucose (Dextran—a polymer of α-(1→6)-linked D-glucose—is synthesized as an EPS by some lactic acid bacteria , 23. Thereported , 25. Dexlinkages . In Leuclinkages , 29, 30.linkages , 32. L.  glucose , 34. By  glucose , 34.http://www.cazy.org/)  (reviewed in (Microbacterium dextranolyticum (formerly Flavobacterium sp. M-73)  was found in the cell surface and culture supernatant of this bacterium more than 40 years ago  randomly hydrolyzes the α-(1→6)-linkages of dextran and produces isomaltooligosaccharides with varying degrees of polymerization. According to the CAZy database (zy.org/) , dextraniewed in ). Dextraiewed in . An enzyp. M-73) . M. dextears ago , 42. Amiears ago . Thus, mFlavobacterium johnsoniae is a Gram-negative soil bacterium that was isolated in 1947. F. johnsoniae belongs to the phylum Bacteroidota and can degrade various polysaccharides, including chitin, and proteins, such as casein and gelatin  and ∼40 PULs . FjGH65A hydrolyzes the α-(1→2)-glucosidic linkage in kojibiose (α-(1→2)-glucobiose) and longer kojioligosaccharides (fjdex31a and fjgh65a are located in the same PUL (FjDexUL), with genes encoding putative proteins involved in carbohydrate utilization; therefore, we hypothesized that FjDexUL is a PUL involved in the utilization of α-(1→2)-branched dextran. Although FjGH65A showed little activity toward α-(1→2)-branched glucan from L. citreum B-1299, it was active against 6-O-α-kojibiosylglucose and 6-O-α-kojitriosylglucose, which are substructures of α-(1→2)-branched dextran, comparable with kojibiose and kojitriose . The expression level of fjdusC dramatically increased (50 fold) when grown on S-32 α-glucan compared with glucose , SusC homolog , SusD homolog , SusE/F homolog , GH66 enzyme , GH31 dextranase , GH97 eentified . To clar glucose . The culn source .Figure 1B. thetaiotaomicron and Thermoanaerobacter pseudethanolicus (TpDex), respectively. In addition, FjGH66 was predicted to be a dextranase because there is no CITase-specific CBM35 domain insertion, which facilitates intramolecular transglycosylation to produce cycloisomaltooligosaccharides, into the catalytic module of FjGH66 (A and B). FjGH66 exhibited superior hydrolytic activity toward linear dextran, and the resultant products comprise glucose, IG2, IG3, IG4, and IG5 (A). FjGH66 degraded IG3, IG4, and IG5 while displaying little hydrolytic activity against IG2, indicating that FjGH66 is a typical endo dextranase (A). FjGH66 showed slight hydrolytic activity toward S-32 α-glucan and S-64 α-glucan and did not hydrolyze soluble starch and pullulan  . FjGH66 f FjGH66  50). Re. ReB. thf FjGH66 , A and B and IG5 A. FjGH66xtranase A. FjGH66pullulan . The kinpullulan .Table 1SC2, and the space group of the IG3 complex is P212121. The overall structures are almost identical (root mean squared deviation of 0.304 < Cα ≤ 0.472 Å); therefore, the following descriptions are based primarily on IG3-complex structure at 1.18 Å resolution. The FjGH66 monomer comprises three domains: N-terminal immunoglobulin fold domain (residues 41–126), (β/α)8-barrel fold catalytic domain (residues 127–464), and C-terminal β-sandwich domain (residues 485–586) (A). A structural homology search using the Dali server revealed that FjGH66 is most similar to TpDex    (A). IG3 occupied the subsites −4 to −2  and the residues forming subsites −3 to +1 were conserved between FjGH66 and GH66 enzymes (C). In the FjGH66 subsite −4, Phe256 and Gly253 were replaced by tryptophan and proline in TpDex, respectively, and Lys429 conformation was different from that of Lys450 of TpDex (C and D). The conformations of Lys429 are identical between the ligand-free and IG3 complex structures (not shown). These may lead to different orientations of Glc −4 between the ligand complex structures of FjGH66 and TpDex (C). In the FjGH66 substrate-binding cleft, O3 of Glc −4 and Glc −3 and O2 of Glc +1 and Glc +2 were exposed to the solvent (C and D). By contrast, subsites −2 and −1 are narrower than the other subsites, and O2 and O3 atoms of Glc −2 and Glc −1 are surrounded by the active site residues (C and D). Therefore, α-(1→6)-linked glucose residues with α-(1→2)- and α-(1→3)-branches would not be accommodated at subsites −2 and −1. However, FjGH66 may be able to hydrolyze the α-(1→6) linkage on the nonreducing end of the α-(1→2)-glucosylated glucose residue in branched dextran due to the space available to accept an α-(1→2)-branch in the vicinity of subsite +1 (D).To elucidate the molecular mechanism of substrate recognition, the crystal structures of FjGH66 in an unliganded form and complexes with glucose, IG2, or IG3 were determined at 1.18 to 1.85 Å resolution . All cry485–586) A. A struy = 40%)  and simiy = 33%)  and dexty = 24%) . The maiy = 24%) A. IG3 ocs et al. ) and the enzymes  and S4.  enzymes C. In theof TpDex , C and Dnd TpDex C. In the solvent , C and Dresidues , C and Dbsite +1 D.Table 3B). FjDex31A produced glucose from the initial stage of the reaction when isomaltooligosaccharides were used as substrates and catalyzed transglycosylation against IG3 and IG4, promoting the formation of longer isomaltooligosaccharides . The activity of FjDex31A against IG3 and IG4 was almost the same, and these specific activities were almost 47- and 3-fold higher than those against IG2 and dextran 40,000, respectively -linked oligosaccharides (p-nitrophenyl α-D-glucopyranoside (pNP-Glc) at pH 7.5 and 30 °C. FjGH97A hydrolyzed α-(1→6)-, α-(1→4)-, α-(1→3)-, and α-(1→2)-linked glucobioses but not trehalose (C and D and C and D). Glucose was the final product of FjGH97A-treated IG3 and IG4, and only glucose production was observed from dextran 40,000 and S-32 α-glucan. These results suggest that FjGH97A is an exo-acting enzyme like other GH97 enzymes. Next, we calculated the kinetics parameters of FjGH97A substrates with α-glucoside linkages  has high hydrolytic activity toward α-(1→6)-glucosidic linkages of dextran, IG2, IG3, and panose and the α-(1→2)-linkage of kojibiose, followed by maltose -branched dextran, comparable with kojibiose and kojitriose . A comparison of the glucose-complex structure (PDB 7FE4 (B). The reducing end of IG2 is directed toward the solvent, whereas 6-OH of Glc +1 at the nonreducing end is blocked by Arg74 (B).FjGH65A showed low activity against S-32 α-glucan and S-64 α-glucan , althougjitriose . To clarexamined . The 1.5examined  and S5. examined A. A compPDB 7FE4 ), where PDB 7FE4 B. The reby Arg74 B.Figure A). Glucose production from S-64 and S-32 α-glucans by a single enzyme was low (<0.21 mM glucose produced). When S-32 α-glucan was treated with FjDex31A, FjGH97A, and FjGH65A, 1.20 mM glucose was produced. Glucose production increased 1.8-fold (2.17 mM) when FjGH66 was included in the reaction (A). Because FjGH66 generates oligosaccharides from S-32 α-glucan, which is a substrate for exo-acting enzymes (FjGH97A and FjGH65A), glucose production increased in the presence of FjGH66. Thus, FjGH66 can release α-(1→2)- and α-(1→3)-glucosylated isomaltooligosaccharides from S-32 α-glucan. The concentration of glucose released from S-32 α-glucan was 21.1% lower when FjGH65A was not included in the reaction. The most significant difference in glucose production was observed in reaction mixtures with or without FjGH97A. The addition of FjGH97A increased the amount of glucose produced from S-32 α-glucan by 1.50 mM (A). Moreover, the amount of glucose produced from S-64 α-glucan was approximately one-half that of S-32 α-glucan in the presence of all FjDexUL GHs (A). This implies that FjDexUL is more proficient in breaking down S-32 α-glucan-type structures than other α-glucans. The degradation products of S-32 α-glucan were analyzed by normal-phase high-performance liquid chromatography (HPLC). The HPLC system can separate glucose, IG2, IG3, IG4, and IG5. The retention times of glucose, IG2, IG3, IG4, and IG5 were 5, 6, 7.5, 9, and 11 min, respectively (B). When S-32 α-glucan was treated with FjGH66, peaks corresponding to glucose, IG2, and IG3 were detected; however, with FjDex31A, peaks of glucose and IG2, as well as a distinct peak at retention time 8.5 min was detected. This peak is likely a minor product of transglycosylation and did not disappear after prolonged incubation or additional enzyme treatment (data not shown). When S-32 α-glucan was treated with all four enzymes, the IG3 peak (produced by FjGH66) disappeared, and the glucose peak was significantly larger. This result suggests that exo-type enzymes play an important role in glucose production from S-32 α-glucan (B). Moreover, endodextranase, α-glucoside hydrolase, and kojibiose hydrolase synergistically degrade S-32 α-glucan into glucose.Based on the substrate specificity of each FjDexUL GH examined, we investigated whether the GHs work together to degrade α-(1→2)- and α-(1→3)-branched dextran. Dextran 200,000, S-64 α-glucan, and S-32 α-glucan were treated with various combinations of FjDexUL GHs A. Glucosreaction A. Becaus 1.50 mM A. MoreovexUL GHs A. This iectively B. When Sα-glucan B. Moreov3CKC), and most likely has one ligand binding site (B. thetaiotaomicron (PDB 6ZAZ), showed that the overall structures are similar (A). However, the conformation of the ligand-binding cleft appeared to vary based on the bound ligand (B–D). The amino acid residues forming the predicted ligand-binding cleft of FjDusD were mostly conserved in closely-related homologs of FjDusD from other bacteria (4FE9) and may have three ligand-binding sites - and α-(1→3)-branched dextran and use FjDexUL-encoded gene products to break down S-32 α-glucan efficiently. The mechanism by which F. johnsoniae is proposed to utilize α-(1→2)- and α-(1→3)-branched α-glucans is shown in C and D). GH13 α-amylases usually hydrolyze α-(1→4) linkages in the vicinity of α-(1→6)-branches in starch with difficulty; however, the catalytic clefts of SusG and Alicyclobacillus sp. 18,711 α-amylase were reported to accept and hydrolyze α-(1→4)-glucan with α-(1→6)-branches  disproportionates maltooligosaccharide length and facilitates further degradation by other GHs. FjGH97A was found to be a novel GH with high activity against isomaltooligosaccharides and nigerose, despite sharing 69% amino acid sequence identity with SusB; the details of its structure–function analysis will be published elsewhere. The IG2-complex structure of FjGH65A suggests that FjGH65A can only hydrolyze the α-(1→2)-glucosidic linkage on the nonreducing end but not on the intermediate α-(1→2)-branches in branched dextran. This is consistent with the fact that FjGH65A shows activity against the α-(1→2)-branched dextran substructures 6-O-α-kojibiosylglucose and 6-O-α-kojitriosylglucose -β-D-Glc-(1→3)-D-Glc and β-D-Glc-(1→4)-β-D-Glc-(1→3)-β-D-Glc-(1→4)-D-Glc, but not for β-D-Glc-(1→3)-β-D-Glc-(1→4)-D-Glc and laminarioligosaccharides , and extracytoplasmic function sigma/anti-sigma factors. These regulators perceive signals by directly interacting with oligosaccharide products through their periplasmic domains. After maltose binds to SusR, the expression of all Sus components is upregulated , 62. Thecharides . The HTCcharides . TherefosusD or mutation in the sugar-binding site of SusD inhibited the growth of B. thetaiotaomicron on starch (B. thetaiotaomicron binds a fructooligosaccharide with a β-(2→1)-linked branch . A f. A fsusDn starch , 65, 66.n starch , 68, 69.d branch  70). To. TosusD Leuconostoc spp. found in wheat sourdough and fermented products and by Lactobacillus sp. from the digestive tract of poultry (M. dextranolyticum (M. dextranolyticum removes α-(1→2) branches extracellularly, degrades dextran, and uptakes isomaltooligosaccharides into the cytosol, whereas F. johnsoniae extracellularly degrades branched dextran to oligosaccharides (with branches) and hydrolyzes all linkages, including branches, in the periplasm. In addition, α-(1→2)- and α-(1→3)-branched dextran exhibited resistance to hydrolysis by digestive enzymes of both human and animal origin -branched dextran and α-glucooligosaccharides, containing α-(1→2)- and α-(1→6)-glucosidic linkages, to produce short-chain fatty acids - and α-(1→3)-branched dextran. Notably, several species of Bacteroides harbor GH97 and GH65 in their putative dextran utilization locus - and α-(1→3)-branches have been reported; the polysaccharide is synthesized as an EPS by  poultry , 71, 72.olyticum . M. dextl origin . Such rety acids , 74, 75.on locus .F. johnsoniae acquired FjDexUL genes to utilize α-(1→2)- and α-(1→3)-branched dextran. Our results will be helpful for understanding complex polysaccharide-mediated interactions between the microbiota in nature. Furthermore, FjDexUL enzymes are expected to be a useful tool for the structural determination of branched dextran.In conclusion, we discovered the PUL targeting EPS from lactic acid bacteria and comprehensively investigated the gene expression and function of proteins present in FjDexUL, demonstrating that FjDexUL recognizes and degrades α-(1→2)- and α-(1→3)-branched dextran. Because bacterial species possessing both GH65 and GH97 genes in dextran utilization loci are limited, it is likely that L. citreum S-64 and L. citreum S-32 were prepared as described  unless otherwise noted. Kojibiose, kojitriose, nigerose, and IG6 were purchased from Carbosynth, and IG2, IG3, IG4, IG5, and inulin were purchased from Tokyo Chemical Industries . α-Glucans from escribed .F. johnsoniae cells were precultured on 0.2% yeast extract and 0.1% MgSO4 and inoculated in 5 ml of 0.2% yeast extract and 0.1% MgSO4 containing 0.5% (w/v) carbohydrates . The cells were cultured for 48 h at 30 °C and harvested by centrifugation . Total RNA was extracted using NucleoSpin RNA plus . DNA was removed by DNase treatment using NucleoSpin RNA Clean-up (Takara Bio), and RNA quality was assessed by calculating absorbance ratios at 260/280 and 260/230 nm using the NanoDrop 2000c (Thermo Fisher Scientific). Then 1 μg RNA was used immediately for reverse transcription with PrimeScript RT Reagent Kit (Takara Bio). The expression of fjdusR, fjdusC, fjdusD, fjdusE, fjdex31a, fjgh66, fjgh97a, and fjgh65a was analyzed using quantitative PCR using THUNDERBIRD SYBR qPCR Mix (Toyobo). The gene-specific primers are listed in https://www.ncbi.nlm.nih.gov/tools/primer-blast/) (-blast/) .F. johnsoniae NBRC 14942 (= ATCC 17061 = UW101) by colony-direct PCR using KOD ONE DNA polymerase (Toyobo) and each pair of primers is listed in E. coli BL21 (DE3) for overexpression. All proteins were produced in 1 L Luria-Bertani medium containing 50 μg/ml kanamycin. Cells were grown at 37 °C with shaking until culture OD600 reached 0.6 to 0.8, at which point isopropyl β-D-thiogalactopyranoside was added at a final concentration of 0.1 mM. Protein expression was induced by overnight shaking at 20 °C. The cells were harvested, resuspended in 50 mM Tris-HCl buffer (pH 7.5) containing 300 mM NaCl and 20 mM imidazole and disrupted by sonication. The cell lysate was centrifuged to remove debris. The supernatant of the lysate was loaded onto a Ni-Sepharose excel  column, and unbound proteins were washed with the same buffer. Proteins were eluted with 50 mM Tris-HCl buffer (pH 7.5) containing 300 mM NaCl and 250 mM imidazole and then concentrated by ultrafiltration using an Amicon Ultra 30,000 molecular cutoff filter (Merck). FjGH97A was further purified by cation exchange chromatography using Mono S 5/50 GL . Equilibration was performed in 20 mM HEPES-NaOH buffer (pH 7.5) and eluted with a linear gradient of 0 to 1 M NaCl. FjGH66 was further purified with anion exchange chromatography using Mono Q 5/50 GL . Equilibration was performed in 20 mM Tris-HCl (pH 7.5) and eluted with a linear gradient of 0 to 1 M NaCl. Protein purity was determined by SDS-PAGE (B). Protein standards  were purchased from Nacalai Tesque. Enzyme concentrations were calculated with ExPASy ProtParam (http://web.expasy.org/protparam/) using molar absorption coefficients  calculated by measuring absorbance at 280 nm with the NanoDrop 2000c (Thermo Fisher Scientific).Recombinant FjDex31A and FjGH65A were expressed and purified as described , 47. TheSDS-PAGE B. ProteiTo evaluate substrate specificity, FjDex31A (100 μg/ml) and FjGH66 (100 μg/ml) were incubated with 10 mM oligosaccharides and α-glucans in 10 mM MES-NaOH buffer (pH 5.5) at 30 °C. FjGH97A (100 μg/ml) was incubated with 10 mM oligosaccharides and α-glucans in Britton–Robinson buffer (pH 7.0) at 30 °C. Reaction mixtures were analyzed by thin-layer chromatography (TLC): 1.5 μl of each sample was spotted on TLC plates of Silica Gel 60 (Merck) and developed with nitromethane:1-propanol:water = 4:10:3. The plates were dried, sprayed with 5% (v/v) sulfuric acid–methanol solution, and charred at 110 °C.2CO3 when nigerose was used as a substrate.To evaluate the specific activities of FjDex31A and FjGH66 against IG2, IG3, IG4, panose, dextran 40,000, dextran 200,000, pullulan, S-64 α-glucan, and S-32 α-glucan were used. The released reducing sugar was quantified using the Somogyi–Nelson method , 78 and To determine if FjDexUL GHs function synergistically, a combination of 100 μg/ml FjDexUL GHs was used to hydrolyze dextran 200,000, S-64 α-glucan, and S-32 α-glucan at 0.5% (w/v) final concentration, pH 5.5, and 30 °C for 10 min or 24 h. The released glucose was determined by the glucose oxidase–peroxidase method and degradation products were detected using HPLC. The reaction mixtures were then applied to a TSK-GEL amide-80 column  immediately after incubation and eluted with 60% (v/v) acetonitrile at a flow rate of 1.0 ml/min at 30 °C. The reaction products were detected using a refractive index detector .7FE3 and the AlphaFold2 model as a search model for the FjGH65A–IG2 complex and FjGH66, respectively  was crystallized as described  and thenectively . Manual ectively , and refectively . Moleculectively . Table 4ectively , 87.N,N′-methylenebisacrylamode = 30:1) and 0.5% (w/v) polysaccharide. FjDusD (7.5 μg), FjDusE (5 μg), and bovine serum albumin (5 μg) were loaded onto gels and electrophoresed under nondenaturing conditions using 0.1 M Tris-HCl (pH 7.8) as the anode buffer and 0.068 M glycine-Tris-HCl (pH 8.9) as the cathode buffer at 80 V for 3 h at room temperature. Proteins were visualized by staining with Coomassie Brilliant Blue.Running gels were composed of 8% polyacrylamide gel  calibrated to 25 °C. All titrations were performed in 10 mM HEPES-NaOH buffer (pH 7.0). Purified FjDusD and FjDusE (0.1 mM each) were loaded into the sample cell, and the syringe was loaded with 5 mM IG5 and IG6 and 10 mM kojibisoe, kojitriose, nigerose, maltose, maltotriose, IG2, IG3, and IG4. An initial injection of 0.2 μl was followed by 19 injections of 2 μl spaced 150 s apart, with an injection duration of 4 s. Titration results were analyzed using MicroCal Origin ITC . Data were fitted to a standard one-site binding model (n = 1), with ligand concentration as a variable. The average and standard error were calculated using data from at least three tests.http://wwpdb.org/) under the accession codes 8IU8, 8IU9, 8IUA, 8IUB, and 8IUC. All other data are contained within the article.The atomic coordinates and structure factors were deposited in the Worldwide Protein Data Bank (This article contains The authors declare that they have no conflicts of interest with the contents of this article.


The low-temperature crystal structures of four organic salts of the anti-spasmodic drug trihexyphenidyl are presented. viz., trihexyphenidylium [1-(3-cyclohexyl-3-hydroxy-3-phenylpropyl)piperidin-1-ium] 4-nitrobenzoate, C20H32NO+·C7H4NO4− (I), trihexyphenidylium 4-hydroxybenzoate, C20H32NO+·C7H5O− (II), trihexyphenidylium 4-bromobenzoate, C20H32NO+·C7H4BrO2− (III), and trihexyphenidylium thiophene-2-carboxylate hemihydrate, 2C20H32NO+·2C5H3O2S−·H2O (IV), conducted at 90 K are described. Structures I, II, and III are solvent free with one cation–anion pair per asymmetric unit, while IV crystallizes as a hemihydrate, having two cation–anion pairs and one water of crystallization in its asymmetric unit. Structures I and III exhibit configurational disorder of the cation. Structure IV also exhibits disorder, but only of the thiophene-2-carboxylate anions. Structure II is a non-merohedric twin by a twofold rotation about [403]. The main supramolecular motifs in I, II, and III are similar R22(10) rings between cation–anion pairs, although their packing within the crystals is distinct. As a consequence of having two cation–anion pairs and a water molecule in its asymmetric unit, the packing in IV is by far the most complex of the four structures, its hydrogen-bonding patterns being quite different from I, II, or III. In all the crystals studied, N—H⋯O, O—H⋯O, and C—H⋯O interactions are observed, plus C—H⋯Br close contacts for III.The syntheses and crystal structure studies of four organic salts of trihexyphenidyl,  It can be used in the treatment of psychotic depression , trihexyphenidylium 4-hydroxybenzoate, C27H37NO4 (II), trihexyphenidylium 4-bromobenzoate, C27H36NO3Br (III) and trihexyphenidylium thiophene-2-carboxylate, which crystallizes as a hemihydrate, C25H35NO3S·0.5H2O (IV).In view of the medicinal importance of trihexyphenidyl, this paper reports the crystal structures of some salts of trihexyphenidyl with organic acids, 2.I, II, III, IV, each trihexyphenidylium cation also includes a chiral carbon, with atoms C1 (C1A and C1B in IV) being the stereogenic centre. Nevertheless, medicinal formulations are racemic, and all four crystal structures :0.497 (4) in I and 0.508 (5):0.492 (5) in III], as shown for I in Fig. 5IV also exhibits disorder, but of the thiophene-2-carboxylate anions [major:minor fractions are 0.795 (2):0.805 (2) and 0.953 (2):0.047 (2) for the inequivalent anion sites]. Structure II is a mon-merohedric twin .Structures The conformations of the trihexyphenidylium cations are determined, in large part, by torsion angles about the C1—C2, C2—C3, C3—N1, C1—C9, and C1—C15 bonds. These are quantified in Table 13.I, II, and III are The main supramolecular motifs in s Figs. 1–3 ▸ ▸, I there are no other strong intermolecular interactions, though there is a weaker C4—H4B⋯O1i  contact between molecules adjacent along the a-axis direction. See Table 2For II, the 4-hydroxy group of the anion is also involved in hydrogen bonding  x, −y, z − In ng Fig. 8. Atom O3III, in addition to the aforementioned ring motif, there are short contacts between C3—H3A and Br1 of a screw-related  anion. Full details are given in Table 4In IV is by far the most complex of the four structures. Its hydrogen-bonding patterns are quite different from I, II, or III. In fact the hydrogen-bonding motifs involving the ‘A–C’ and ‘B–D’ cation–anion pairs are themselves distinct. For the ‘A’ cation, the N1A—H1NA group is an asymmetric bifurcated hydrogen-bond donor to O1C and O2C . The hydroxyl group of cation A forms a hydrogen bond (as donor) to O2C of a translation-related  anion. These combine to form A–C cation–anion chains that extend parallel to the a-axis  anion. The net result of these hydrogen bonds are cation–anion–water chains that also propagate along the a-axis direction -tricyclamol iodide, which has an N-methyl-pyrrolidinium ring.A search within the Cambridge Structural Database  for I, and in methanol (5 ml) and acetonitrile (5 ml) for 4-hydroxybenzoic acid  (II), 4-bromobenzoic acid  (III) and thiophene-2-carboxylic acid  (IV). The resulting solutions were stirred for 30 minutes at 333 K and allowed to stand at room temperature. X-ray quality crystals were formed on slow evaporation over the course of a week for all of the compounds. The melting points were 409–411 K (I), 425–427 K (II), 395–396 K and (III) 368–369 K (IV).A solution of commercially available trihexyphenidyl   in methanol (10 ml) was mixed with equimolar solutions of the appropriate acids in methanol (10 ml) 6.sp2—H), 0.99 Å (R2CH2), and 1.00 Å (R3CH) using Uiso(H) values constrained to 1.2Ueq of the attached carbon atom. Cation disorder in I and III was modelled using similar combinations of restraints  and constraints (SHELXL command EADP). Disorder of the thiophene-2-carboxylate anions in IV corresponded to a ∼180° flip of the thiophene ring, which is common for thiophene, and was modelled using geometry restraints (SAME and FLAT) and displacement parameter constraints (EADP). Structure II was twinned by non-merohedry, corresponding to a twofold rotation about the real-space direction [403]. Diffraction data were integrated using two orientation matrices and scaled/merged following standard procedures .Crystal data, data collection, and refinement statistics are given in Table 610.1107/S2056989023005960/vm2286sup1.cifCrystal structure: contains datablock(s) I, II, III, IV, global. DOI: 10.1107/S2056989023005960/vm2286Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989023005960/vm2286IIsup3.hklStructure factors: contains datablock(s) II. DOI: 10.1107/S2056989023005960/vm2286IIIsup4.hklStructure factors: contains datablock(s) III. DOI: 10.1107/S2056989023005960/vm2286IVsup5.hklStructure factors: contains datablock(s) IV. DOI: Click here for additional data file.10.1107/S2056989023005960/vm2286Isup6.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989023005960/vm2286IIsup7.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989023005960/vm2286IIIsup8.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989023005960/vm2286IVsup9.cmlSupporting information file. DOI: 2280174, 2280173, 2280172, 2280171CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


However, the biological roles of these metabolites were unknown. Therefore, we aimed to investigate the inflammatory and mesenchymal-epithelial transition (MET) effects of 1-nonadecene, and the inflammatory and collagen precipitation effects of l-lactic acid on both periodontal ligament fibroblasts (PdLFs) and peripheral blood mononuclear cells (PBMCs). PdLFs and PBMCs were treated with 1-nonadecene and l-lactic acid. Cytokines’ expression was measured using quantitative real-time polymerase chain reaction (qRT-PCR). E-cadherin, N-cadherin, and macrophage polarization markers were measured using flow cytometry. The collagen, matrix metalloproteinase (MMP)-1, and released cytokines were measured using collagen assay, western blot, and Luminex assay, respectively. In PdLFs, 1-nonadecene enhances inflammation through the upregulation of some inflammatory cytokines including IL-1β, IL-6, IL-12A, monocyte chemoattractant protein (MCP)-1, and platelet-derived growth factor (PDGF) α. 1-Nonadecene also induced MET through the upregulation of E-cadherin and the downregulation of N-cadherin in PdLFs. 1-Nonadecene polarized macrophages to a pro-inflammatory phenotype and suppressed their cytokines’ release. l-lactic acid exerted a differential impact on the inflammation and proliferation markers. Intriguingly, l-lactic acid induced fibrosis-like effects by enhancing collagen synthesis, while inhibiting MMP-1 release in PdLFs. These results provide a deeper understanding of 1-nonadecene and l-lactic acid’s roles in modulating the microenvironment of the periapical area. Consequently, further clinical investigation can be employed for target therapy.Recently, 1-nonadecene and  Metabolism and metabolites represent a major contributor in modulation of such microenvironment2. Recently, we have identified 1-nonadecene and l-lactic acid as the highest unique metabolites in radicular cysts and periapical granulomas, respectively3. The contribution of l-lactic acid in the formation of facial granulomatous tissue after poly-l-lactic acid injection was previously reported4. However, its mechanism of pathogenesis or contribution in other tissues have never been tested. On the other hand, 1-nonadecene was biologically identified for the first time in radicular cysts3. 1-Nonadecene is a natural metabolite identified in bacteria5 and fungi6. There are, however, insufficient data available on its biological and immunological activities in human cells. Therefore, we aimed to investigate the immunological and metabolic effects of these two metabolites on both PdLFs and macrophages. PdLFs represent the most predominant cells within the periapical area that play a critical role in the remodeling process and wound healing7. Furthermore, the periapical area was predominantly infiltrated with lymphocytes, plasma cells, and macrophages8.Microenvironment  of a lesion plays important roles in its initiation and pathogenicity9. These epithelial cells are expected to arise from the epithelial rests of Malassez10, while a recent study reported the possible conversion of dermal fibroblasts to epithelial cells11. However, the underlying mechanism is not reported. On the other hand, periapical granulomas consist of an organized profuse collagen fibers in diverse directions appearing as irregular dense connective tissue with vascular elements12.The inner layer of radicular cyst cavity is composed of stratified squamous epithelium with an outer wall of dense fibrous capsule infiltrated with chronic inflammatory cells13. The main reparative function of PdLFs is to secrete extracellular matrix components such as collagen, that builds up the periodontal ligament and its fibers14. Besides, macrophages are important components of the inflammatory process15 in response to microbial infiltration in the periapical area and hence shaping up a specific periapical lesion16. Microbial metabolites are more predominantly contributing to such inflammatory conditions17.PdLFs can act as immune cells and secret different cytokines3. Radicular cysts and periapical granulomas are among the most frequently occurring pathological lesions in the alveolar bone, accounting for 95% of the periapical radiolucency18. Interestingly, a radicular cyst may evolve from a previous periapical granuloma19, indicating a metabolic change in the lesion microenvironments for the induction of each lesion3. Thus, understanding the inflammatory and immunological roles of the major unique metabolites in the initiation of such lesions may provide potential diagnostic and therapeutic values.In response to microbial entrance to the apical area, the host mounts a series of immunological and metabolic reactions that result in the destruction of periapical tissue and the development of cysts and granulomasl-lactic acid on PdLFs and PBMCs were investigated as follows:The effects of different concentrations of 1-nonadecene and The gene expression of IL-1α, IL-1β, IL-6, IL12A, MMP-1, MCP-1, transforming growth factor (TGF)β1, PDGFα, and vascular endothelial growth factor (VEGF) α were investigated in PdLFs and PBMCs following the treatment by 1-nonadecene. In PdLFs, 1-nonadecene caused significant upregulation at 1 µM and 10 µM for IL-1α after 2- and 6-day treatments  (P˂0.0001), while all concentrations significantly caused the upregulation of classical/HLA-DR/CD86, and classical/CD86 (P˂0.0001)  and for classical/CD206 at 10 µM and 100 µM  was not observed at all concentrations of 1-nonadecene but the activated form of non-classical/HLA-DR/CD86 at 1 µM and 10 µM (P˂0.0001)     Fig. E, while 01) Fig. F. MCP-1 01) Fig. G,H. For 01) Fig. I,J, whil01) Fig. K and at 01) Fig. L. In PBM01) Fig. M–R.Figurl-lactic acid at 1 µM and 100 µM caused significant upregulation in the expression of gene coding collagen  after 2-day treatment, while 10 µM caused similar upregulation of only COL3α1 , 10 µM (P = 0.001), and 100 µM (P = 0.01)  Fig. G, while 01) Fig. H.Figure P˂0.0001) for 2 days   after 6-day treatment  Fig. C,D and aent Fig. E,F. The l-lactic acid caused significant polarization to classical phenotypes (CD14high/CD16low), classical/HLA-DR, classical/HLA-DR/CD86, classical/CD86, and classical/CD163 (P˂0.0001)  /HLA-DR, non-classical/HLA-DR/CD86, and non-classical/CD86 were significantly upregulated at all concentrations of l-lactic acid, while the non-classical/CD163 was significantly upregulated at 1 µM and 10 µM l-lactic acid (P˂0.0001)  , but downregulation in PBMCs. MCP-1 showed both patterns of upregulation and downregulation at different concentrations, time points33, and cell types34. Additionally, some studies showed a proliferative effect of lactic acid through the elevated levels of VEGFα35 and PDGFα36 in macrophages, while in our study, these markers were downregulated in PBMCs. On the other hand, we observed both upregulation and downregulation of VEGFα and PDGFα in PdLFs. These results were expected since we have used different concentrations and times for the treatment . Since 1-nonadecene has not been studied before in any experiment, we selected the same concentrations of l-lactic acid. PdLFs were incubated at 37 °C and 5% CO2 for 2 days and 6 days when treated with l-lactic acid45. For 1-nonadecene, the treatment regimen followed a clinical study regarding the MET phenomenon46 with some modifications, including the daily treatment of the PdLFs with fresh media containing 1-nonadecene. For MET induction in 1-nonadecene treatment, 2-day and 6-day treatments were used to check the irreversibility of MET induction47. Phorbol 12-myristate 13-acetate (PMA)  at 1 µM48 was used as an inflammatory inducer49, a positive control for the expression and synthesis of collagen50, MMP-151, N-cadherin52, and as a negative control for the expression of E-cadherin54.Human PdLFs  were cultured in Dulbecco's Modified Eagle medium/nutrient mixture F-12 (DMEM/F-12)  supplemented with 10% fetal bovine serum (FBS)  and 1.5% penicillin–streptomycin . PdLFs were treated in triplicates with 1 µM, 10 µM, and 100 µM 1-nonadecene  and 55. PBMCs were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium  supplemented with 10% FBS and 1.5% penicillin–streptomycin. PBMCs were treated in triplicates with 1 µM, 10 µM, and 100 µM 1-nonadecene and l-lactic acid. These concentrations were chosen to be compatible with the concentrations used in the treatment of PdLFs. For better differentiation of the PBMCs, the incubation period was extended to 7 days56. Lipopolysaccharide (LPS)  at 100 ng/ml57 was used as a positive control for M1 polarization57 and 15 ng/ml IL-458  was used as a positive control for M2 polarization54.Fresh PBMCs were isolated using histopaque gradient separation  according to the manufacturer’s instructions and Soliman et al. 202059 using QuantStudio RT-PCR . IL-1α, IL-1β20, IL-6, IL12A3, MMP-160, MCP-161, TGFβ129, PDGF α62, and VEGF α63 are differentially expressed in radicular cysts and correlated to epithelial cells’ proliferation. IL-1α, IL-1β64, IL-63, MCP-165, PDGFα66, and VEGFα67 are differentially expressed in periapical granulomas. Primers of IL-1α, IL-1β, IL-6, IL-12A, MMP-1, MCP-1, TGFβ1, PDGFα, VEGFα, E-cadherin, N-cadherin, collagen (COL)1α1, COL3α1, and COL5α1 were used to study their gene expression in PdLFs and/or PBMCs. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as housekeeping gene for normalization to other target genes and the relative fold change was calculated using 2^−(ΔΔCt). The sequences of the employed primers are described in Supplementary Table PdLFs, and PBMCs were used to quantify gene expression. RNAs from the lysate of cells were extracted using RNeasy Mini Kit , then reverse transcribed to cDNA using SuperScript™ III first-strand synthesis system  according to the manufacturer’s instructions. qRT-PCR setup and cycling procedures were done as previously describedPdLFs were treated with 1 µm, 10 µm or 100 µm 1-nonadecene, in addition to 1 µm PMA. The cells were harvested and fixed in stain wash buffer (SWB)  and the cells were then treated with rabbit anti-human E-cadherin Alexa 647  or rabbit anti-human N-cadherin  according to the manufacturer's instructions. Secondary antibody goat anti rabbit IgG Alexa 488 was used for N-cadherin . Corresponding isotype controls were used to compare the expression of the markers.l-lactic acid, 100 ng/ml LPS, and 15 ng/ml IL-4. Harvested cells were treated with the anti-CD14-APC-Cy7, anti-CD16-Alexa 700, anti-HLA-DR-PE-Cy7, anti-CD86-PE, anti-CD163-PerCP-Cy5.5, and anti-CD206-APC  in SWB and incubated according to the manufacturer's instructions. The stained cells were fixed in fixation and permeabilization solution . PdLFs and PBMCs were then acquired in the BD FACSAria III flow cytometer  using FACSDiva software with standard configuration. Compensation for PBMCs was performed by using BD CompBeads. Analysis and characterization of the markers were performed using BD FlowJo software . The gating strategies was performed as previously described68.For PBMCs, the cells were treated with 1 µM, 10 µM or 100 µM 1-nonadecene, l-lactic acid, 100 ng/ml LPS, and 15 ng/ml IL-4. PBMCs were treated in triplicate for human cytokines detection using a magnetic bead-based multiplex assay  of the Luminex platform as per the manufacturer’s instructions. A total of 12 cytokines were assessed in this study including those known in M1 polarization such as IL-1β, IL-12p70, CXCL9, CXCL10, IL-2369, tumor necrosis factor (TNF)-α70, and IFN-γ71. IL-10, IL-1372, CCL1773, and CCL2270 were used for M2 polarization and IL-674 as a pleotropic cytokine. The fluorescence of the beads was measured using a Luminex BioPlex 200 analyzer , and the data analysis was performed using BioPlex manager software .Cytokine assay was performed on the supernatant of treated PBMCs with 1 µM, 10 µM or 100 µM 1-nonadecene, 6 PdLFs treated with 1 µM, 10 µM, or 100 µM l-lactic acid in comparison to 1 µM PMA for 2 and 6 days. The experiment was conducted in triplicates, and the manufacturer’s instructions were followed.Soluble collagen assay kit  was used to detect the collagen formation in 1 × 10l-lactic acid compared to 1 µM PMA for 2 and 6 days and the western blot was performed according to Hamoudi et al.75. The cell pellets were lysed in RIPA buffer  supplemented with 1:10 protease and phosphatase inhibitor cocktail-EDTA free . The protein lysates were quantified using the Pierce BCA protein assay kit . 10 µg Protein samples were used for MMP-1 detection using MMP-1  rabbit monoclonal antibody and normalized with β-actin (13E5) rabbit monoclonal antibody according to the manufacturer’s instructions. The blots were visualized using the Clarity Western ECL Substrate  in the ChemiDoc Touch Gel and Western Blot Imaging System . Image Lab Software (V 6.1.0) was used to detect and quantify the protein bands.To investigate the protein expression of MMP-1 in PdLFs, the cells were treated with 1 µM, 10 µM or 100 µM P-value < 0.05 was considered significant. * Reveals that P-value < 0.05, ** reveals that P-value < 0.01, *** reveals that P-value < 0.001, **** reveals that P-value < 0.0001.Statistical analysis was performed using GraphPad Prism software (version 9.1.0). Data were analyzed by one-way and two-way analysis of variance (ANOVA) using Dunnett's multiple comparisons tests as indicated per each graph. Supplementary Information.