Moreover, cytotoxicity of the compounds was evaluated in vitro against two cancer cell lines including a human melanoma (A375), a human colon adenocarcinoma (HT29), and also one normal cell human foreskin fibroblast (HFF). The selective and potent cytotoxicity effect was exhibited by C1 and C3 on cancer cell lines. The apoptosis through a caspase-dependent mitochondrion pathway was confirmed by ROS production, MMP reduction, p53 activation, Bax up-regulation, and Bcl-2 down-regulation, cytochrome c release, procaspase-9, and 3 expression, for A375 cells treated to C1 and C3. According to similar cellular uptake of the complexes in A375 cell line, the generation of ROS was considered as an effective agent to justify the inhibition effect C1 and C3 on mentioned cells. Furthermore, arresting the cell cycle in the G2-M phase and inducing apoptosis were indicated by these two complexes.Three novel Tl(III) complexes (C1), (C2) and (C3) were synthesized using the one-pot reactions of pyridine dicarboxylic acid derivatives, 2-aminobenzimidazole and/or 4-aminopyridine, and also thallium(III) nitrate trihydrate metal salt. The structure of all three complexes was determined by the single-crystal X-ray diffraction. C1 and C2 were realized to be isostructural with disordered square anti-prismatic geometry and for C3 arrangement of the distorted tricapped triangular prism was proposed. Cyclic voltammetry measurements on the complexes exhibited that formal potential values are more positive for C1 (E The studies have shown that many metal ions from these groups can be effective in the hydrolysis of peptides and proteins and even some proteolytic enzymes require such ions for their activities2. Among them, metal ions of group 13 have gained a special position due to potential applications in the treatment and diagnosis of diseases3. Thallium is the latest metal introduced in this group and because of high toxicity, poor investigations have been performed on it4. Thallium can be found in the environment in two oxidation states of Tl(I) and Tl(III) though the main form is Tl(I). Thallium in this oxidation state represents a high similarity to potassium cation5. The toxicity of Tl(I) is related to its interference in the vital potassium-dependent processes so that it can substitute potassium in the (Na+-K+)-ATPase and represent a high affinity for sulfhydryl groups from proteins and other biomolecules.The unique chemical and biological properties of main-group elements have put them in the spotlight201Tl has been applied in imaging of the myocardium and tumors7 and indicated achievements for recognizing the proliferation of small lung cancer4.From the point of medicine, thallium has been reported effective in the treatment of diseases such as syphilis, tuberculosis, and malaria. Such effectiveness is related to the high toxicity of this metal. On the other hand, the isotope8. The evaluations on the toxicity rate of thallium show that coordinating with some organic molecules has a significant role in the reduction of its toxicity rate9. So that some of the thallium(III) porphyrins can represent favorable medical applications1. Accordingly, M. Mimouni et al., in 201410 designed thallium complexes from monensin and lasalocid and evaluated their antibacterial, antifungal, and antitubercular activities. In this study, favorable pharmaceutical applications without a high risk of toxicity were recognized for the complexes11. Also, antimicrobial applications have been proven in some thallium compounds such as Tl acetate and Tl carbonate6. In another study, the anti-tumor activity of para-substituted tetraphenyl porphyrin Tl(III) salicylate complexes was evaluated and a strong inhibition effect (up to 90%) of 5-SSATl(III)t(4-OCH3)PP was exhibited against all four human cancer cell lines tested including MCF-7, THP-1, PC-3 and A5491. In 2013, A. S. El-Tabl et al., synthesized Tl(I) complexes from 2-(2-(4-carboxyphenyl)guanidino)acetic acid as ligand and exhibited a potent anti-proliferative effect on the MCF-7 cell line compared to the standard drug12. Accordingly, new hopes emerged for the continuation of biological studies on thallium compounds13. But, besides medicine fields, thallium and its compounds have indicated applications as semiconductors, catalysts, dyes, and pigments14. Such diverse uses led to consider thallium compounds in various areas of knowledge15.The chemical properties of Tl(I) such as the presence of lone electron pair, large size, salts that are often anhydrous, diverse coordination numbers, and geometries have caused that, despite high toxicity, its coordination compounds be especially regardedSPS:refid::bib. It is a very important topic in biochemistry because the oxidation state of the metal has a significant role in the biological properties of inorganic compounds17. For example, thallium(III) has a higher toxicity than Tl(I)18. But besides the role of metal and its oxidation state, the nature of ligand coordination should not be ignored to evaluate the cytotoxicity effects of metallic complexes. It has been proven that versatile anionic ligands including carboxylate groups with diversity in the coordination modes are usually favorable options to design metal complexes and an assay of their medicine activity8. In 2005, M. Rafizadeh et al., synthesized the thallium(I) complex containing 2,6-Pyridinedicarboxylic acid (pydcH2) and reported its crystal structure19. Investigations have indicated that among various diacids, pyridine-2,6-dicarboxylic acid20 has the most favorable biochemical and chemical features21.Some studies exhibited that organic ligands can reduce Tl(III) to Tl(I) Therefore in the present study, three novel thallium complexes were designed from pyridine dicarboxylic acid derivatives and were characterized by single-crystal X-ray diffraction. In the next step, the electrochemical and anticancer properties of the compounds and as well as the correlation between these two activities were evaluated. Then, detailed studies were performed to recognize the apoptosis pathway.P-1 space group. These two complexes were isostructural and contain anionic units [Tl(pydc)2(H2O)2]− for C1 and [Tl(hypydc)2(H2O)2]− for C2. Also, extranuclear cations (2-abH)+ and water molecules co-crystallized are observed along with complex units in these structures. In the anionic parts of both C1 and C2 respectively, (pydc2−) and (hpydc2−) molecules coordinated to one Tl(III) atom, through two oxygen and one nitrogen atoms, and two water molecules were also available in the coordination sphere +. The strongest hydrogen bonds seem to be N(2)–H(2)···O(11)  and O(5 W)–H(5WB)···O(8 W)  for C1; and O(5B)–H(5B)···O(1 W) and O(12 W)–H(12A)···O(2D)  for C2. These interactions lead to the appearance of cyclic hydrogen bonding synthons as RRRRRR24.Hydrogen bonds containing O–H···O, N–H···O and C–H···O with D···A distances ranging from 2.63(14) to 3.49(15) Å for C1  and 2.64(14) to 3.39(12) Å for C2  lead to the formation the supramolecular networks in these compounds+ at distance 3.18(11) Å, and angle 87.8(2)° . These interactions were observed for C2 between N(2C)–H(2C) with  and C(1B)–O(2B) with (2-abH)+ at distances 3.28(11) and 3.50(11) Å, and angles 82.8(2) and 84.5(3) °, respectively .Besides hydrogens bonding, stacking interactions containing π-π, C–O···π and C–H···π are also shown in these structures which help to form more connections between anionic and cationic moieties and subsequently grow the crystal. C–O···π interactions occur for C1 between C(1D)–O(2D) and C(1B)–O(2B) respectively, with , and  rings at distances 3.26(11) and 3.13(12) Å, and angles 91.6(2) and 93.7(3)°, and also, C(7D)–O(4D) with (2-abH)P21/c space group with four moieties in the unit cell. In the structure C3, one anionic unit [Tl(pydc)(pydcH)2]−, a (4-apyH)+ cationic moiety, and five water molecules uncoordinated were observed. In this compound, three pyridine-2,6-dicarboxylate molecules coordinate to Tl(III) in tridentate mode through two oxygen atoms and one nitrogen atom so a prism with three caps of three nitrogen atoms on its faces is exhibited and distorted tricapped triangular prism geometry is suggested for Tl(III) nine coordinated. According to the best of our knowledge and data obtained from the Cambridge crystallographic data centre, nine coordination number has not been observed for Tl(III) complexes synthesized of pyridine dicarboxylic acid derivatives until now26 and it is reported for the first time by us. In this structure, the difference of bond length C7A–O3A = 1.20(13) and C7B–O3B = 1.20(13) with C7A–O4A = 1.30 (13) and C7B–O4B = 1.30(13) confirmed the presence of COOH groups.Compound C3 and the coordination polyhedron around Tl(III) atom is shown in Fig. x + 1, y, z], O(4 W)–H(4WB)···O(2A) and O(4 W)–H(4WA)···O(4C)  for C3. The presence of water molecules in this structure increases the probability of the formation of hydrogen bonds and also has a significant role to create water clusters, three-dimensional networks, and following the stability of the crystal lattice. The views of (H2O)n tetrameric and pentameric water clusters are shown for C3 in Figures S7 and S827.The sum of bond angles, N1B–Tl1–N1C = 109.9 (7), N1A–Tl1–N1B = 116.6 (7) and N1A–Tl1–N1C = 130.5 (7) ° is exactly equal to 357.1° which proves that N1A, N1B, and N1C atoms form a planar triangle around Tl(III). Also, two other triangles are observed from three atoms containing O1A, O1B, and O1C, and three atoms containing O3A, O3B, and O3C, on two sides of the first triangle . The angles between oxygens indicate that they are eclipsed. The intra- and intermolecular hydrogen bonds include N–H···O and C–H···O with D···A distances ranging from 2.72(13) to 3.52(14) Å for this structure. The strongest hydrogen bonds can be N(4D)–H(4D)···O(2C) [–1. The voltammograms are exhibited in Fig. 0ˊ 0.109, –0.051, and 0.244 V were obtained for C1, C2, and C3, respectively. The electrochemical oxidation of all three complexes seems be a one-stage process which includes irreversible couples with EC = –0.363 V, EA = 0.472 V for C1, EC = –0.271 V, EA = 0.220 V for C2 and EC = –0.337 V, EA = 0.581 V for C3. The peaks can be interpreted according to the Tl(III)/Tl(I) redox system for complexes9. As can be seen, the oxidation potential of C1 and C3 is at more positive values compared to C2. The investigations indicated that ligands do not have remarkable electrochemical reactivity. Therefore, redox couples are related to only the metal centers in complexes28.The electrochemical behavior of the complexes and also their ligands was studied by the cyclic voltammograms in the potential range of + 1.5 to –1.5 V and a scan rate of 50 Vs2O . Then the inhibitory effect of the compounds was evaluated by MTT assay against A375, HT29, and HFF cell lines  compared to oxaliplatin  while cytotoxic effect for C2  was indicated weaker than C1 and oxaliplatin on these cells. Here, the results confirmed that C3  has the most significant effect of inhibition compared to other compounds on A375 cells. Also, the most cytotoxicity on HT29 cell line is exhibited by C3 . On the other hand, all three complexes indicated the weak cytotoxicity toward human foreskin fibroblast cells . The potent inhibitory effect on A375 cells merits C1 and C3 for detailed evaluations. According to the best of our knowledge, reports on the anticancer effect of thallium complexes are rare and there are limited studies in this field. In this study, since thallium is located in the center of all three complexes, and also ligands coordinated to thallium are pyridine dicarboxylic acid derivatives, the obtained results can highlight the structure − activity relationship. Paying attention to these points provides useful information for designing more potent and selective pharmaceutical compounds with fewer side effects in the field of cancer treatment.In this study, A375 cells exhibited higher sensitivity to C1 3.3H2O and C1–C3 to investigate whether the type of ligand and coordination geometry can be effective on cellular uptake. A375 cells treated with all three complexes for 24 h exhibited a remarkable increase in the cellular concentration of thallium compared with Tl(NO3)3—treated cells. The results of this evaluation clearly showed that weak cytotoxicity C2 compared to C1 and C3 is related to its inherent properties since all complexes are drawn easily into the cells . When we evaluated cellular uptake of C3 in HFF cells , it was realized that the cellular concentration of thallium was less in normal cells than cancer cells (A375), but the amount of thallium in these cells was not zero; therefore it cannot be concluded with certainty that the reason for the low efficacy of the C3 against normal cells is only due to its low penetration into these cells.The cellular uptake of pharmaceutical compounds has usually an important role in the rate of their biological activityThese results directed our study toward the determination of ROS in A375 and HFF cells following treatment with the complexes.36. In our study, all three complexes selected to investigate intracellular ROS produced in A375 cells and data analyzed by flow cytometry after 24 h .The high-value generation of reactive oxygen species (ROS) is among the most effective agents on the imbalance of cellular redox homeostasis and is followed by cell destruction. Therefore, compounds with the ability to produce ROS have a prominent role in cellular activities. In some anticancer studies, it has been recognized that inhibition properties of the compounds on cancer cells have affected the rate of ROS generated by them38. In this study, the effect of C1 and C3 on cell cycle distribution in A375 cells was evaluated to distinguish whether these complexes can exert their cytotoxic activity through alteration in cell cycle progression and/or induction of apoptosis in mentioned cells. As results indicated, both C1 (16.91%) and C3 (21.10%) were able to induce a significant rise in the cellular population of the G2-M phase in A375 cells .Cell cycle analysis was performed by flow cytometry to identify the percentage of time that cells spend in different phases of the cell cycleboth C1 1.91% and 38. As shown in Fig. 50 concentrations for 24 h, induced 14.22 and 11.47% early apoptosis and, 48.01 and 50.19% late apoptosis while these values were 0.13 and 1.27% in the control group, respectively. Quantification of the results shows that the C1 and C3 are 62.24 and 61.66% more effective than vehicle control-treated .Investigations have indicated that the formation of the tumor has been usually detected following mutations in mitochondrial genes and the generation of intracellular reactive oxygen species (ROS)ial Fig. a; while ial Fig. b,c38,39.44. It has been realized that Bcl-2 family proteins have a prominent role in the intrinsic mitochondrial signal pathway and are strongly related to apoptotic cell death. As can be seen in Fig. To reveal the apoptotic pathway activated by the complexes, the expression levels of several anti-apoptotic and pro-apoptotic proteins were analyzed by Western blot in A375 cells. The aforementioned proteins play effective roles in inhibiting/inducing cell growth and apoptosis. In this study, both C1 and C3 significantly enhanced the level of p53. Additionally, the expression of p53 activates the regulation of Bcl-family proteins45. So, in the present study, to perform further evaluations on the apoptotic pathways of cell death, the effects of the synthetic complexes were investigated on the expression of procaspases 9, and 3. The results confirmed that the level of procaspases 9 and 3 generally increased in the complexes-treated A375 cells  porphyrin complexes were presented48. According to the reports, it was realized that thallium can form various organic and inorganic compounds with both oxidation states (I) and (III)6, and oxidation state (II) was observed in organometallic complexes of thallium though this state was the least seen49.For the first time, thallium complexes were synthesized using protoporphyrins by Fischer and Pu¨tzer in 1929. In the next studies, the ability of thallium(III) salts to form complexes from neutral ligands was evaluated30. Here, electrochemical studies confirmed that these ligands, despite having no electrochemical activity, can affect the electrochemical activity of the complexes. So, the presence of only OH group in the 4-position of the pyridine ring of C2 caused that, despite having the same metal center, geometry, and counter ions, C1 and C2 to be completely different in their formal potentials. Also, the electrochemical behavior of C3 was different from C1. Such difference may be related to various geometries in these two complexes. Therefore, coordination geometry can also be an important factor in the redox activity of the complexes so that the formal potential for C3 is exhibited two and five times higher than C1 and C2, respectively. In MTT assay, it was exhibited that C1 has more potent cytotoxicity than C2 toward A375 cells. According to crystallographic data, the only difference between structures C1 and C2 was related to the presence of OH in the 4-position of the pyridine ring of C2. It seems that this group led to decreases in cytotoxicity C2 compared to C1. Therefore, it can be concluded that the type of ligand coordinated and substitutions on it has a high impact on the anti-tumor activities of inorganic compounds. Also, the stronger anti-proliferative effect of C3 compared to C1 can be related to the difference in coordination geometry and extranuclear cation in these complexes (with the same ligand coordinated).In our study, three new Tl(III) complexes with pyridine dicarboxylic acid derivatives were reported. Many studies have proved that the pyridine dicarboxylates family as a ligand can be suitable options to design diversity of metal complexes50. The results obtained from our electrochemical evaluations following previous studies in this field show that C1 and C3 with further formal potential E0ˊ(0.109 V) and (0.244 V) respectively exhibit higher inhibitory effects compared to C2 (–0.051) on the cancer cell lines. Such a feature can arise from the effectiveness of these compounds on the cellular redox homeostasis of cancer cells through the production of excessive values of ROS51. Many studies have proven that some anti-cancer compounds can interfere with the metabolic balance of ROS and enhance its level. This increase can lead to the collapse of mitochondrial membrane potential, an increase of the mitochondrial outer membrane permeability, and finally apoptosis38. To better understand why C1 and C3 are more cytotoxic than C2 against A375 cells, before ROS assay, cellular uptake of Tl in these cells was evaluated. The results indicated that all three complexes can penetrate easily into the A375 cells. Therefore, intracellular ROS amount was measured and the results were exhibited that ROS value generated by C2 is very lower than the other two complexes. The presence of OH group in C2 was considered as an important agent on the decrease of electrochemical activity as well as ROS production by this complex. Although the toxicity mechanism of Tl(III) is still unknown, the investigations have somewhat confirmed that its cytotoxicity may be due to creating oxidative stress states. Oxidative stress can destruct proteins, lipids, carbohydrates, and nucleotides and also, induce mutations to the DNA. Oxidative DNA damage has a remarkable role in the pathophysiology of numerous diseases, including cancer, atherosclerosis, and neurodegenerative disorder9. The study of operating mechanisms of thallium in various cell lines, tissues, as well as distinct animal species has proven that in most cases, the formation of ROS was the main factor for tissue damage and organ dysfunction52. In this study, the data obtained from cell cycle analysis and apoptosis assay confirmed that C1 and C3 could arrest cells in the G2-M phase and induce apoptotic cell death. The fluorescence microscope images indicated that these two complexes can decrease the mitochondrial membrane potential in A375 cells. Mitochondria have an important role in most eukaryotic cells to produce energy and regulate apoptosis. It was concluded that in most cases, the generation of intracellular ROS can lead to cellular damage especially the collapse of mitochondria membrane potential. The produced ROS impair the electron transport chain and lead to apoptosis39. Following results reported in this field, the decrease of MMP leads to the release of pro-apoptotic proteins which start the death-signaling cascade. Cytochrome c is a small hemeprotein found loosely associated with the inner membrane of the mitochondrion that can activate the caspase cascade and lead to apoptotic cell death. In our study, the expression of procaspases 9 and 3 increased in the complexes-treated A375 cells following the release of hemeprotein mentioned. Also, mitochondrial outer membrane permeabilization was proved through a mechanism involving Bcl-2 family proteins which comprises anti-apoptotic Bcl-2, and Bcl-xl, and pro-apoptotic Bax, Bim, and Bad54. In this line, p53 is a key protein in controlling cell responses to DNA damage via cell cycle arrest and apoptosis, which increases the level of responses to oxidative stress44. Here, p53 activation, Bax up-regulation, and Bcl-2 down-regulation, procaspase-9, and 3 expression proved that anticancer activity of C1 and C3 is through inducing mitochondria-mediated apoptosis  complex containing pyridine dicarboxylic acid derivatives were synthesized and their structures were determined by X-ray crystallography. Despite structural similarities, different redox couples were exhibited in electrochemical studies for the complexes. C1 and C3 indicated stronger electrochemical activity compared to C2. Also, the potent cytotoxicity effect was showed toward cancer cell lines by these two complexes. The presence of the OH group in the 4-position of the pyridine ring of C2 was considered as an effective agent on the decrease of electrochemical and anticancer properties of it. Here a direct correlation was observed between the anti-proliferative effect and the formal potential of the compounds. Since cellular uptake assay was indicated similar permeation for all three complexes in A375 cells, it seems that active redox compounds can be more effective on ROS generation and induction of apoptosis. In further evaluations, the release of cytochrome c and the increase of procaspases 9 and 3 expression were observed in A375 cells treated by C1 and C3. Therefore apoptosis through the caspase-dependent mitochondrion pathway was proposed as the main mechanism for cell death. On the other hand, our evaluations indicated that C3 can penetrate in HFF cells and generate ROS, but ROS produced either because of the low amount or its weak effectiveness in normal cells does not hurt normal cells compared to cancer cells.1HNMR spectra were obtained from Bruker Ultrashield 400 spectrometer. Microanalyses  were measured with a Perkin-Elmer 2004(II) elemental analyzer. Electrochemical experiments were performed using a µAUTOLAB modular electrochemical system  equipped with a PGSTAT type III module driven by GPES software in conjunction with a conventional three-electrode system an Ag/AgCl/3 M KCl and platinum wire as reference and counter electrode respectively and a GC as the working electrode.Pyridine-2,6-dicarboxylic acid, 4-hydroxy-pyridine-2,6-dicarboxylic acid, 2- aminobenzimidazole, 4-aminopyridine, and thallium(III) nitrate trihydrate were purchased from the commercial sources and were used as received. The solvents were distilled for all synthetic works. Melting points were obtained on an Electrothermal IA-9100 apparatus. The FT-IR spectra were recorded on a Bruker Vector 22 FT-IR spectrometer using KBr pellets. Electronic spectra were recorded on Specord 210, Analytik Jena spectrophotometer in the range of 200–900 nm at room temperature. The 3)3.3H2O . The solution was stirred for 3 h at room temperature. Yellow transparent crystals were obtained by slow solvent evaporation after 14 days. Yield (78%). M.p: 340 °C. Found : C 32.67 (32.81), H 3.32 (3.25), N 9.17 (9.11)%. δH : 8.36 , 7.40 , 7.19 , 3.38. Selected IR bands : 3454, 3344 (νNH2), 1658 s (νCO). UV–Vis: λmax , 271 .An aqueous solution of pyridine-2,6-dicarboxylic acid  and 2- aminobenzimidazole  solved in methanol were simultaneously added to a stirring aqueous solution of Tl was used in place of pyridine-2,6-dicarboxylic acid. Finally, the solution was stirred for 3 h at room temperature. Colorless transparent crystals were obtained by slow solvent evaporation after 30 days. Yield (84%). M.p: 356 °C. Found : C 29.79 (29.82), H 3.48 (3.55), N 8.24 (8.28)%. δH : 8.38 , 8.02 , 3.41 . Selected IR bands : 3458, 3338 (νNH2), 1656 s (νCO). UV–Vis: λmax , 269 .This complex was synthesized by a method identical to C26H28N5O17Tl): C 35.24 (35.17), H 3.09 (3.15), N 7.76 (7.89)%. δH : 8.36 , 7.96 , 7.16 , 3.51 . Selected IR bands : 3485, 3367 (νNH2), 1664 s (νCO). UV–Vis: λmax , 297 .This complex was prepared by a method identical to C1, but 4-aminopyridine  in water was used in place 2-aminobenzimidazole. Then, the unclear solution was refluxed for 5 h at 100 °C. The white precipitate was filtered off and recrystallized from methanol. White crystals were collected after 36 days. Yield (69%). M.p: 248 °C. Found . Cells were seeded into a 96-well plate and incubated for 24 h at 37 °C, 5% CO2, and 95% air. Then the medium was replaced with the respective medium containing ligands or Tl(III) complexes at different concentrations and incubated for 48 h. 10 μL of MTT was added and the medium was removed after 4 h of incubation. Finally, the violet crystal was solubilized in 100 μL DMSO and the absorbance was measured at 570 nm. IC50 value was measured by the standard method. The evaluations were carried out in triplicate23.In this study, A375 (a human melanoma), HT29 (a human colon adenocarcinoma), and HFF (a human foreskin fibroblast) cell lines were provided from the Pasteur Institute (Iran). The cells were cultured as recommended at 37 °C in a humidified atmosphere of 5% CO7 cells) were treated with 10 μM C1–C3 and Tl(NO3)3.3H2O, respectively, for 24 h at 37 °C, 5% CO2, and 95% air. Cold PBS  was used for rinsing the cells three times after being collected. Cells were lysed in 1 M NaOH (1 mL) and diluted with 2% (v/v) HNO3 (5 mL) to determine whole-cell thallium content. The amount of thallium taken up by the cells was measured by ICP-MS. The instrument calibrates thallium using a standard solution containing 10, 50, 100, 500, and 1000 ppb thallium. This test was performed as explained on HFF cells treatment to C338.A375 cells (1 × 106 cells/mL) were induced with C1–C3 (at IC50 concentrations) at 37 °C for 24 h, serum-free medium washed twice. Then, the cells were incubated in 2 mL of serum-free medium containing H2DCF-DA (2 μM) for 30 min at 37 °C, serum-free medium washed twice. Cells were collected and assayed by flow cytometer with excitation wavelength at 489 nm and emission wavelength at 524 nm, and ten thousand events were collected. The DCF fluorescence peak for this cell sample was evaluated and data were analyzed by FlowJo software. This test was performed as explained on HFF cells treatment to C3 (at the highest concentration)38.The generation of intracellular ROS was evaluated by reactive oxygen species assay kit. 2 mL of A375 cells  and resuspended in 0.5 mL of PBS containing 100 μg/mL DNase-free RNase A , 50 μg/mL propidium iodide  in the dark for 30 min. The distribution of the cells in different phases of the cell cycle was determined using flow cytometry  and FlowJo software v10.6.1 (https://www.flowjo.com/solutions/flowjo/downloads) was used to analyze the data. This test was performed as explained on HFF cells treatment to C3 (at the highest concentration)62.A375 cells were seeded in 6-well plates with 1.5 × 1050 concentrations. After 24 h of incubation, cells were washed with PBS, trypsinized, centrifuged, and re-suspended in 100 μL binding buffer at a concentration of 1 × 106 cells/mL, then added 5 μL of annexin V-FITC conjugate (30 min), and 5 μL of Propidium Iodide (PI) to each cell suspension (20 min) in the dark. The samples were analyzed using a flow cytometer , and the results were assessed using the FlowJo software62.Annexin V-FITC and PI staining (Annexin V/PI) apoptosis detection kit were used for assessing the percentage of dead cells by apoptosis pathway. Briefly, A375 cells were treated with C1 and C3 at IC5 cells/mL) were treated with IC50 concentrations of C1 and C3 in 6-well plates for 12 h. Cells were harvested and washed three times with PBS after 24 h of incubation. Then cells were stained with 1 mL of JC-1 stock solution (10 mg/mL). Assays were initiated by incubating A375 cells with JC-1 for 30 min at 37 °C in the dark and detected with a fluorescence microscope. This test was performed as explained on HFF cells treatment to C3 (at the highest concentration).Mitochondrial membrane potential analysis was determined by fluorescent dye JC-1 staining according to the manufacturer's protocol for the JC-1 assay kit. In brief, 2 mL of A375 cells , and then placed on ice for 15 min. Total proteins were extracted by centrifuging the cell sample at 10,000 rpm and 4 °C for 10 min. Then about 50 μg of purified proteins were mixed with an equal volume of electrophoresis sample buffer, and the mixture was heated for 10 min. Meanwhile, the equal protein content from the control cells was prepared in the same way. The cell sample (10 μL) was loaded onto 10% SDS-PAGE gels and then transferred onto a microporous polyvinylidene difluoride (PVDF) membrane. The membrane was blocked with 5% BSA in TBST buffer for more than 2 h. After the removal of TBST buffer, membranes were incubated with an appropriate dilution of specific primary antibodies in TBST overnight at 4 °C and then washed with TBST three times. The membranes were incubated with secondary antibodies for 1 h and washed with TBST. The immunoreactive signals were detected using an enhanced chemo-luminance kit (Pierce ECL Western Blotting Substrate) following the procedures given in the user manual63.After incubation with C1 and C3 at ICSupplementary Information.


C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontal temporal degeneration (FTD), which is attributed to both a gain and loss of function. C9orf72 forms a complex with SMCR8 and WDR41, which was reported to have GTPase activating protein activity toward ARF proteins, RAB8A, and RAB11A. We determined the cryo-EM structure of ARF1-GDP-BeF3- bound to C9orf72:SMCR8:WDR41. The SMCR8longin and C9orf72longin domains form the binding pocket for ARF1. One face of the C9orf72longin domain holds ARF1 in place, while the SMCR8longin positions the catalytic finger Arg147 in the ARF1 active site. Mutations in interfacial residues of ARF1 and C9orf72 reduced or eliminated GAP activity. RAB8A GAP required ~10-fold higher concentrations of the C9orf72 complex than for ARF1. These data support a specific function for the C9orf72 complex as an ARF GAP. The structure also provides a model for the active forms of the longin domain GAPs of FLCN and NPRL2 that regulate the Rag GTPases of the mTORC1 pathway.Mutation of  C9orf72:SMCR8:WDR41 complex has been reported to have GAP activity for both ARF family proteins and the RAB proteins RAB8A and RAB11A. Here the authors provide structural and biochemical evidence for a specific function of the C9orf72 complex as an ARF GAP, and a structural framework for the GAP activity of the longin-containing GAP family. C9ORF72 is the most prevalent genetic cause of two clinically overlapping neurodegenerative diseases, amyotrophic lateral sclerosis (ALS) and frontal temporal degeneration (FTD)2. There are two mutually non-exclusive mechanisms for C9ORF72 expansion-induced pathogenesis in ALS/FTD, gain of function from the presence of toxic RNA and dipeptide repeats4, and loss of function due to reduced C9orf72 protein expression7. The reduction or absence of C9orf72 has been proposed to alter vesicle trafficking10, autophagy13, lysosomal homeostasis16, actin dynamics17 and inflammation18. However, the direct biochemical function(s) of C9orf72 have been uncertain until recently.Expansion of GGGGCC repeats in the first intron of 15. Both C9orf72 and its cellular binding partner SMCR8 are longin and DENN domain  containing proteins19. Two cryo-electron microscopy structural studies of the C9orf72:SMCR8:WDR41 complex found that SMCR8 is sandwiched between C9orf72 and WDR4121. Structural similarity was observed between C9orf72:SMCR8 and another double longin-DENN complex, the folliculin and folliculin-interacting protein 2 (FLCN-FNIP2) complex23. FLCN-FNIP2 is a GTPase activating protein (GAP) complex for RagC in the mTORC1 signaling pathway, with the putative catalytic Arg residue residing in the FLCN subunit. This suggested that C9orf72:SMCR8 might also be a GAP for some small GTPase21. The C9orf72 complex was reported to have GAP activity for both ARF family proteins21, and the RAB proteins RAB8A and RAB11A20. The conserved Arg147 of SMCR8 was proposed to serve as the catalytic Arg finger typical of most GAP proteins. Mutation of SMCR8 Arg147 eliminated GAP activity, consistent with the Arg finger hypothesis. However, in the absence of a structure determination for the active state of any of these complexes, there is not yet a consensus in the field with respect to the Arg finger mechanism24.C9orf72 exists in cells as a stable heterotrimer with Smith–Magenis chromosome regions 8 (SMCR8) and WD repeat-containing protein 41 (WDR41)25, as does C9orf7226. ARF6 and RAB11 vesicles28 have been implicated in retrograde vesicle transport in axons, such that excessive ARF6GTP and RAB11GTP have been proposed to inhibit anterograde transport and axonal regeneration29. Nevertheless, the reported dual specificity of the C9orf72 complex is unusual. ARF and RAB proteins belong to distinct small GTPase families. Yet C9orf72 is inactive as a GAP against the close ARF relatives ARL8A and B, and also inactive against RAB1A and 7A21. Thus, the C9orf72 complex is not simply a promiscuous GAP. In order to clarify the specificity of the C9orf72 complex GAP activity, we sought to determine structures of the complexes with representative ARF in GAP-competent states. We selected ARF1 as a structurally well-characterized model for the ARF family, having previously found that GAP activity against ARF1, 5, and 6 was indistinguishable21. We found that the GAP reaction went to completion at an order of magnitude lower C9orf72 complex concentration for ARF1 as compared to RAB8A (greater than tenfold). Consistent with a preference for ARF proteins, it was possible to determine the structure of the ARF1:C9orf72:SMCR8:WDR41 complex in the presence of the GTP hydrolysis ground-state mimetic BeF3−. These data validate that the ARFs are specific substrates of C9orf72 complex GAP activity. The structure provides a model for the catalytic geometry of the larger longin-domain-based GAP family, including the FLCN:FNIP2 and GATOR1 complexes.The discovery that the C9orf72 complex has GAP activity against subsets of both ARF and RAB proteins was exciting in that both of these small GTPase families participate extensively in membrane traffic21. The concentration of ARF1 was fixed at 30 µM, and then treated with 0, 0.3, 0.6, and 3-µM C9orf72 complex for 15 min at 37 °C, and nucleotide products were monitored by high pressure liquid chromatogram (HPLC) analysis. The 0.3 µM C9orf72:SMCR8:WDR41 sample, equivalent to a 1/100 molar ratio relative to ARF1, was sufficient to completely hydrolyze all of the GTP-bound to ARF1  and then tested if they could still promote the GTP hydrolysis for the purified ARF1. In the presence of BeF3−, but not AlFx−, GAP activity was inhibited. Thus, we generated a nucleotide hydrolysis ground-state analog31 by incubating the GDP-bound protein complex with BeF3− overnight, and visualized it by cryo-EM. A preliminary reconstruction of C9orf72:ARF1-SMCR8:WDR41 was obtained from a dataset collected on a 200-kV Talos Arctica. This reconstruction showed extra density corresponding to ARF1 localized atop the two longin domains 30 33  were identified. This family of ARF activators comprises at least 15 members36, which are the targets of the relatively unspecific natural product brefeldin A. A number of highly specific ARF GEF inhibitors have been identified, and strategies for their screening are well worked out44, lending support to the plausibility of such an approach.The working hypothesis suggested by the data above is that C9orf72 haploinsufficiency in ALS and FTD results in an excess of ARFThe gene encoding ARF1 (residues E17-K181) was amplified and inserted into a pCAG vector encoding an N-terminal STREP-STREP-FLAG tagged SMCR8 using KpnI restriction sites  was added to 50-mL hybridoma media (Invitrogen) and 1 mg of total DNA (isolated from transformed E. coli XL10-gold) in another 50-mL hybridoma media. Each 1 mg of transfection DNA contained an equal mass ratio of the three C9orf72 complex subunit expression plasmids. PEI was added to the DNA, mixed, and incubated for 15 min at room temperature. One hundred milliliters of the transfection mix were then added to each 1-L culture. Cells were harvested after 3 days. Cells were lysed by gentle rocking in lysis buffer containing 50-mM HEPES, pH 7.4, 200-mM NaCl, 2-mM MgCl2, 1% (vol/vol) Triton X-100, 0.5-mM TCEP, protease inhibitors  and supplemented with phosphatase inhibitors (50-mM NaF and 10-mM beta-glycerophosphate) at 4 °C. Lysates were clarified by centrifugation  and incubated with 5-mL glutathione Sepharose 4B  for 1.5 h at 4 °C with gentle shaking. The glutathione Sepharose 4B matrix was applied to a gravity column, washed with 100-mL wash buffer , and purified complexes were eluted with 40-mL wash buffer containing 50 mM reduced glutathione. Eluted complexes were treated with TEV protease at 4 °C overnight. TEV-treated complexes were purified to homogeneity by injection on Superose 6 Increased 10/300 GL  column that was pre-equilibrated in gel-filtration buffer . For long-term storage, fractions from the gel-filtration chromatography were snap-frozen in liquid nitrogen and kept at −80 °C.HEK293-GnTi cells adapted for suspension were grown in Freestyle media supplemented with 1% FBS and 1% antibiotic-antimycotic at 37 °C, 80% humidity, 5% CO6-tagged ARF1 (residues E17-K181) and all the ARF1 mutants, the plasmids were transformed into E. coli BL21 DE3 star cells and induced with 1-mM IPTG for 3 h. The cells were lysed in 50-mM Tris-HCl pH 8.0, 300-mM NaCl, 2-mM MgCl2, 10-mM imidazole, 0.5-mM TCEP, and 1-mM PMSF by ultrasonication. The lysate was centrifuged at 15,000 × g for 30 min. The supernatant was loaded into Ni-NTA resin and washed with 20 and 50-mM imidazole and eluted with 300-mM imidazole. The eluate was further purified on a Superdex 75 10/300 GL  column equilibrated in 20-mM HEPES, pH 7.4, 200-mM NaCl, 2-mM MgCl2, and 0.5-mM TCEP. STREP-STREP-FLAG tag RAB8A (residues M1-S181) was expressed in HEK293-GnTi cells and purified by Strep resin and eluted in 10-mM desthiobiotin buffer. The eluted protein was applied on Superdex 75 10/300 GL column. The nucleotide-binding state of each small GTP-binding protein was assessed by HPLC (see below). The purified RAB8A was GDP-bound and our previous data demonstrated that it had spontaneous guanine exchange activity21, therefore RAB8A was incubated with GTP at room temperature for 2 h and applied to a PD 10 column to remove the excess nucleotide. The resulting RAB8A sample had roughly 45% GTP-bound. ARF1I49H/G50R and ARF1I49H/G50R/K73W were also GDP-bound, therefore the GTP reloading protocol was also applied to these samples.For expression of HisQ71L complex, 15 μM of C9orf72:SMCR8:WDR41 was incubated with 15-fold of ARF1Q71L in 500-μl overnight and injected into Superose 6 Increased 10/300 column.For the assembly of C9orf72:SMCR8:WDR41 with ARF1The proteins used in this study are shown in Supplementary Fig. 2, and 0.5-mM TCEP, and incubated with BeF3− overnight. 3.5 μL of sample was deposited onto freshly glow-discharged  C-flat 1.2/1.3-3Au grids (Electron Microscopy Sciences), and blotted for 4 s using a Vitrobot Mark IV (FEI) with 30-s incubation, blot force 8 and 100% humidity and temperature set to 22 °C. The complex was visualized in the Talos Arctica operated at an acceleration voltage of 200 kV, and equipped with a Gatan K3 Summit director electron detector in super-resolution counting mode at 36,000×, corresponding to a pixel size of 0.57 Å. In total, 2579 movies were acquired with the defocus range between −1 and −2.5 μm with a 3 × 3 image shift pattern prior to stage movement. All movies consisted of 50 frames with a total dose of 50 e−/Å2 and a total exposure time of 5.16 s.For the Talos Arctica dataset, the purified complex was diluted to 0.16–0.2 mg/ml in 20-mM HEPES pH 7.4, 200-mM NaCl, 2-mM MgCl−/Å2 and a total exposure time of 2.929 s.For the untilted Titan Krios dataset, grids were prepared in the same manner and the complex was collected using a Titan Krios G2 electron microscope (FEI) operating at 300 kV with a Gatan Quantum energy filter (operated at 20-eV slit width) using a K3 summit direct electron detector  in super-resolution counting mode at magnification 81,000×, corresponding to a pixel size of 0.47 Å on the specimen level. In total, 10,188 movies were collected with defocus of −1.2 to −2.5 μm with a 3 × 3 image shift pattern prior to stage movement. Movies consisted of 50 frames, with a total dose of 50 e−/Å2 for 5.326 s at CDS mode.The Titan Krios dataset with a stage tilt of 30° was obtained using 4-μL sample applied on freshly glow-discharged  UltrAufoil R1.2/1.3 grid (Ted Pella) and plunged into liquid ethane after 30-s incubation and blotting for 4 s using a Vitrobot Mark IV (FEI) with blot force 0 and 100% humidity and temperature set to 5 °C. Four thousand five hundred and eighty-five movies were collected using SerialEM at around −1.5–2.8-μm defocus with 1 × 3 image shift pattern. The movies were exposed to a total dose of 50 e−/Å2 for 5.326 s at CDS mode.The Titan Krios dataset with a stage tilt of 40° was collected using 4 μL sample applied on freshly glow-discharged  GF-1.2/1.3-3Au-45nm-50 grid (Electron Microscopy Sciences) and plunged into liquid ethane after 30-s incubation and blotting for 3 s using a Vitrobot Mark IV (FEI) with blot force 8 and 100% humidity and temperature set to 4 °C. Six thousand one hundred and seventy-eight movies were collected using SerialEM at −1.5–2.8-μm defocus with 1 × 3 image shift pattern. The movies were exposed to a total dose of 50 eAll of the data were acquired with SerialEM using custom macros for automated single particle data acquisition. Imaging parameters for the dataset are summarized in Supplementary Table 45 and using 7 × 5 patches and binned 2x by Fourier cropping to a calibrated pixel size of 1.14 Å/pixel. The defocus values were estimated by CTFFIND446 from summed imaged without dose weighting. After manual check to remove empty or crystalline ice micrographs, 251 micrographs were discarded and the remaining 2328 micrographs were subjected to reference-free particle picking using gautomatch-v0.53 (http://www.mrc-lmb.cam.ac.uk/kzhang/Gautomatch/). About four million particles were extracted with binning 4x in Relion 3.147 and applied to iterations of three-dimensional (3D) classifications. The best class was selected and re-extracted with binning 1x. The particles at the edges were removed and imported into cryoSPARC v248 for further two-dimensional (2D) classification clean up and non-uniform refinement. The resulting 80,569 particles were refined to 4.84 Å and used as reference map for further Titan Krios dataset processing. The workflow is depicted in Supplementary Fig. For the Talos Arctica dataset, drift and beam-induced motion correction was applied on the super-resolution movie stacks using MotionCor2http://www.mrc-lmb.cam.ac.uk/kzhang/Gautomatch/) and then extracted with binning 6x. The particles were subjected to 3D classification using a 45-Å low-pass filtered reference generated by the reconstruction collected in Talos Arctica microscope. 888,231 particles from the best class were selected and refined to 7.755 Å. The refined particles were further cleaned up with rounds of 2D and 3D classification. The extracted, unbinned particles was refined to 4.5 Å and applied to CTF refinement for beam-tilt and per-particle defocus determination as well as Bayesian polishing. The shiny particles were further refined to 3.7 Å. Map sharpening was performed with DeepEMhancer49 using the “tightTarget” or “highRes” models. The workflow is depicted in Supplementary Fig. For the untilted Krios dataset, drift and beam-induced motion correction was applied on the super-resolution movie stacks using MotionCor2 using 7 × 5 patches and binned 2x to a calibrated pixel size of 0.94 Å/pixel. The defocus values were estimated by CTFFIND4 from summed imaged without dose weighting. Other processing procedures were performed within Relion 3.1. Seven thousand three hundred and forty-seven micrographs with CtfMaxResolution values better than 5 were selected for further processing. Around six millions of particles were picked using gautomatch-v0.53 . Particle orientation distributions were plotted using scripts (https://github.com/fuzikt/starpy). 3DFSC curve for the reconstruction was calculated on the 3DFSC processing server51.All reported resolutions are based on the gold-standard FSC 0.143 criterion. cryoSPARC v2 database files were converted to Relion star file using the UCSF PyEM package script , and mouse ARF1 (PDB: 1O3Y)32 were rigid body fitted separately into the density map using UCSF Chimera52. The Leu71 of ARF1 was mutated to its wild-type residue Gln and the GTP was replaced as GDP-BeF3−. Atomic coordinates were refined by iteratively performing Phenix real-space refinement and manual building and correction of the refined coordinates in Coot. To avoid overfitting, the map weight was set to 1 and secondary structure restraints were applied during automated real-space refinement. Model quality was assessed using MolProbity53 and the map-vs-model FSC g. The supernatant was loaded onto a HPLC column . Nucleotides were eluted with HPLC buffer  at flow rate 0.6 ml/min. The identity of the nucleotides was compared to GDP and GTP standards.The nucleotides bound to small GTPases were assessed by denaturing 30 μl of protein to 95 °C for 5 min followed by 5-min centrifugation at 16,000 × g. The supernatant was injected onto an HPLC column as described above. The experiments are carried out at least three times and one representative plot is shown.HPLC-based GTPase assays were carried out by incubating 30 μl of 30-μM GTPases with or without GAP complex at a 1:100, 1:50, or 1:10 molar ratio as indicated for 15 min at 37 °C. Samples were immediately boiled for at least 5 min at 95 °C and centrifuged for 5 min at 16,000 Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


Habenaria linearifolia and Habenaria cruciformis in South Korea. Overall, the genetic diversity was well maintained in South Korean Habenaria, but high FST values were estimated suggesting large population diversification with limited gene flow. Bayesian assignment analysis revealed a morphologically cryptic diversity pattern in Jeju Island populations, which might serve as an evolutionarily significant unit.Monitoring intraspecific diversity offers invaluable insights on conservation practices as the variation is the product of species evolution. Accordingly, the role of population genetic diversity has drawn great attention over the last century responding to the biodiversity loss induced by a series of anthropogenic changes. Orchids are one of the most diverse, yet ironically most rapidly disappearing plant groups due to the specialized habitat preferences. Thus, population-level genetic diversity studies may offer a powerful tool for orchid conservation programs. Using the 3 restriction site-associated DNA (3RAD) approach, 2,734 genome-wide single nucleotide polymorphisms (SNPs) were isolated. With the 2,734 SNPs, we investigated genetic diversity and population structure on 72 individuals of  Plagiorhegma dubium from the Red List of South Korea based on the pattern of population genetic diversity of simple sequence repeat data despite the recent growth of census population sizes.Monitoring the population-level genetic diversity for a species offers invaluable insights into conservation practices as the variation directly reflects the evolutionary and ecological process that the species has experienced . BiodiveOrchidaceae is a cosmopolitan plant group that comprises well beyond 20,000 taxa exhibiting the most remarkable diversity of all levels from infraspecific to generic . IronicaHabenaria Willd. is one of the most diverse taxonomic groups of the terrestrial orchids distributed across the old and the new worlds, which consists of more than 800 taxa  Szlachetko, a rare terrestrial orchid with a narrow distribution  is restricted to bogs and wet grasslands  markers, we aimed to (1) assess the extent of genetic diversity within and among populations; (2) evaluate gene flow among H. linearifolia populations and between the two species; (3) determine the presence of genetic bottlenecks; and finally (4) identify any cryptic diversity that is presently unknown. Given the specialized habitat preferences and the obligatory symbiotic relationships with pollinators and mycorrhizal fungi, we hypothesized that the populations of H. linearifolia have largely differentiated with low within-population genetic diversity. Habenaria cruciformis population might have largely diverged from all H. linearifolia populations with very limited gene flow if the species is an independent species with complete reproductive isolation. The results may suggest separate management units and/or assist in determining the conservation priority.In this study, we surveyed genomic diversity and the spatial pattern of Habenaria linearifolia is 25–90 cm tall with whitish-green flowers blooming from July to September . Index adapters  were ligated in 15 μl reactions containing 100 ng DNA, 8 μl master mix, and 1 μl of each 5 μM adapter. We prepared the master mix to make sure each 15 μl reaction volume contained 1 reaction of each restriction enzyme and 1.5 μl 10× FastDigest Buffer. The digestion and ligation were administered simultaneously. We then incubated the samples in a thermal cycler for 15 min at 37°C. The ligation mixture was prepared as follows: 0.5 μl 10× Ligase Buffer, 100 units T4 DNA ligase, 1.5 μl 10 mM ATP, and 2 μl ultra-pure water. We added 5 μl ligase mixture to each sample and subsequently incubated at 22°C for 20 min and 37°C for 10 min. The process was repeated two times, then incubated at 80°C for another 20 min. We ran a test PCR to check the ligation results using Bioneer multiplex premix. A 20 μl reaction was made containing 1 μl each of iTru5 and iTru7 primers and 1 μl of adaptor-ligated DNA fragments. Samples were amplified using the following thermal cycler profile: 95°C for 10 min; 35 cycles of 95°C for 30 s, 60°C for 1 min, 72°C for 30 s; and 72°C for 5 min. We then pooled 5 μl of the adaptor-ligated fragments from each sample in a 1.5 ml tube. Pooled samples were purified using a 1:1.8 mixture of AmPure XP magnetic beads . We washed the samples with 70% EtOH, which then sat at room temperature for 15 min to air dry and resuspended with 60 μl TE buffer. Subsequently, a 50 μl reaction was prepared containing 10 μl of the pooled DNA fragments, 5 μl of 5 μM iTru5 primer, 5 μl of 5 μM iTru7 primer, 10 μl 5× HF Buffer, 1.5 μl of 10 μM dNTP, and 1 unit of Phusion DNA polymerase. We then amplified the samples: 98°C for 2 min; 7 cycles of 98°C for 20 s, 60°C for 15 s, 72°C for 30 s; and 72°C for 5 min. Another sample clean-up was administered by using a 1:1.8 mixture of AmPure beads, washed twice with 70% EtOH and resuspended in 60 μl TE. For the targeted size selection of 500-bp fragments (± 10%), we used Pippin Prep . Final amplification was conducted using 50 μl PCRs containing 5 μl of size-selected DNA, 3 μl of 5 μM P5 primer, 3 μl of 5 μM P7 primer, 1.5 μl of 10 μM dNTP, 10 μl 5× HF Buffer, and 1 unit of Phusion DNA Polymerase. The thermal cycler profile was as follows: 98°C for 2 min, 98°C for 20 s, 61°C for 15 s, and 72°C for 45 s for 12 cycles; and 72°C for 5 min. We did a final clean-up for the amplicons with a 1:1.8 mixture of AmPure beads, washed twice with 70% EtOH and resuspended in 35 μl ultra-pure water. We evaluated the completed 3RAD library for quality and quantity in Agilent 2100 Bioanalyzer . We ran the library on an Illumina HiSeq X-10 using 2 × 150 paired-end sequencing at Macrogen Inc., South Korea.For genotyping, we employed 3RAD , which ide novo due to the lack of a fully assembled and annotated reference genome. To assemble catalogs from the quality-filtered reads, we set the parameters as −m 3 and −M 3 in the ustacks function, then we allowed a maximum of one mismatch between sample loci  following p 5 and −r 0.80). We further screened the SNP loci that significantly departed from the Hardy–Weinberg Equilibrium , expected heterozygosity (He), observed heterozygosity (Ho), and percentage of polymorphic loci were computed in GenAlex 6.5  and Euclidean distances  by GenAlex and Bayesian model-based clustering by STRUCTURE v. 2.3.4 . For theH. linearifolia and one cluster of H. cruciformis. For the analysis, we randomly selected 303 unlinked SNPs to ease the computational complications. We utilized a coalescent-based approach implemented in MIGRATE- N 3.6.11  to 0.30 , H. cruciformis started exhibiting a unique ancestry pattern with a minor portion of admixture  were greater than the ones observed in many rare orchids [Spiranthes spiralis, mean FST = 0.02 , 2,734 SNPs have the eleven populations of H. linearifolia and H. cruciformis assigned to two demes consisting of the inland deme and the Jeju Island deme  that is currently shrinking with the anthropogenic interferences  BioProject database under accession number S-RL and S-YJ conceived the research idea and designed the experiments. S-YJ got the funding and revised the manuscript. T-YC conducted library preparation and related laboratory work. S-RL and T-YC performed genetic and statistical analyses. S-RL wrote the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Nr2e3rd7/rd7 mice, a mouse model of the recessively inherited retinal degeneration enhanced S-cone sensitivity syndrome (ESCS). We characterized in detail C57BL/6J Nr2e3rd7/rd7 mice in vivo by fundus photography, optical coherence tomography and fluorescein angiography and, post mortem, by histology and immunohistochemistry. White retinal spots and so-called ‘rosettes’ first appear at postnatal day (P) 12 in the dorsal retina and reach maximal expansion at P21. The highest density in ‘rosettes’ is observed within a region located between 100 and 350 µM from the optic nerve head. ‘Rosettes’ disappear between 9 to 12 months. Non-apoptotic cell death markers are detected during the slow photoreceptor degeneration, at a rate of an approximately 3% reduction of outer nuclear layer thickness per month, as observed from 7 to 31 months of age. In vivo analysis of Nr2e3rd7/rd7 Cx3cr1gfp/+ retinas identified microglial cells within ‘rosettes’ from P21 on. Subretinal macrophages were observed in vivo and by confocal microscopy earliest in 12-months-old Nr2e3rd7/rd7 retinas. At P21, S-opsin expression and the number of S-opsin expressing dorsal cones was increased. The dorso-ventral M-cone gradient was present in Nr2e3rd7/rd7 retinas, but M-opsin expression and M-opsin expressing cones were decreased. Retinal vasculature was normal.The photoreceptor-specific nuclear receptor Nr2e3 is not expressed in  Nr2e3rd7/rd7  mouse is a mouse model of the recessively inherited enhanced short-wavelength (S)-cone sensitivity syndrome , caused by bi-allelic pathogenic variants in NR2E3 (nuclear receptor class 2 family E member 3). The expression of the photoreceptor-specific nuclear receptor Nr2e3 is abolished in Nr2e3rd7/rd7 mice because of an L1 retrotransposon inserted into exon 5: the Nr2e3 intron 5 is retained and incompletely spliced isoforms accumulate in the photoreceptor nuclei, preventing protein translation1. In wild-type mice, Nr2e3 expression is uniquely restricted to rod photoreceptors in the mature retina4. The rod precursors are generated over an extended developmental period, starting at embryonic day (E) 13 in the central retina, peaking around birth and ongoing until postnatal day (P) 66. In the absence of Nr2e3, the early-born post-mitotic photoreceptor precursors that are normally committed to the rod fate become ‘blue’ S-cones7, yielding in a two to threefold increase in ultrastructurally normal S-cones in Nr2e3rd7/rd7 retinas10. Because the physiological function of Nr2e3 is to repress cone-specific gene expression in rods, a subset of cone genes is derepressed in Nr2e3rd7/rd7 rods11, resulting in a hybrid photoreceptor expressing rod and cone genes, coined ‘cod’10. Derepressed genes comprise those of the cone-specific phototransduction pathway12, and genes allowing the ‘cods’ to access the cone-specific visual cycle, as demonstrated by a markedly accelerated dark-adaptation13. The ultrastructure of the ‘cods’ is intermediate between normal rods and cones, with an approximately 30% larger cell body than normal rods, and an increased quantity of euchromatin and juxtanuclear mitochondria that is characteristic of cones10.The rd7 phenotype was initially identified on an albino-like 77–2C2a background and then backcrossed into a pigmented C57BL/6J background16. At 1 month of age, Nr2e3rd7/rd7 mice showed white spots all over the retina and waves, whorls and rosette-like structures  in the retinal outer nuclear layer16. At 5 months of age the white spots had nearly disappeared and waves, whorls and ‘rosettes’ had flattened out16. By 16 months of age the white spots were completely gone, as well as the retinal folding. The ablation of cones by mating Nr2e3rd7/rd7 mice with a mouse line carrying a transgene directing cone-specific expression of the diphtheria toxin chain A, was sufficient to abolish retinal folds and ‘rosette’ formation17. The hyperautofluorescent material localized within the ‘rosettes’ was shown to be due to macrophage infiltration and/or presence of microglial cells, presumably because the retinal pigment epithelium (RPE) alone is unable to remove the waste of photoreceptor outer segments within the ‘rosettes’19.The autosomal recessive rd7/rd7 mice, and discuss the findings with respect to the potential disease mechanisms underlying ESCS.Here we analyzed in detail the progression of retinal degeneration in pigmented C57BL/6J Nr2e3Nr2e3rd7/rd7 retinas during early postnatal development, we performed fundus examination and optical coherence tomography (OCT). White spots appeared first dorsally at postnatal day 12 (P12) in C57BL/6J Nr2e3rd7/rd7 retinas .To further describe and quantify the early retinal phenotypes of C57BL/6J ogy Fig. . At P12,ogy Fig. B. At P13ogy Fig. D. The nuNr2e3rd7/rd7 retinas at P16, P21 and P28 to detect the microglial cell marker Iba1  and the F4/80 antigen preferentially expressed by murine mononuclear blood cells and macrophages  microglia was localized to the inner and outer plexiform layers, as well as the nerve fiber layer  monocytes/macrophages were present in retinal and choroidal vessels. In C57BL/6J Nr2e3rd7/rd7 retinas, Iba1+ microglia exhibited an ‘activated’ shape at P16, identified by larger cell bodies and thicker processes  in microglial cells under the control of the endogenous Cx3cr1 locus. We observed a decreased overall number of ‘rosettes’ in this Nr2e3rd7/rd7 Cx3cr1gfp/+ mouse line of mixed genetic background in comparison to the C57BL/6J background (data not shown). Fundus fluorescence imaging readily detected GFP+ retinal microglial cells . By 15 months of age ONL thickness was further reduced by 21.7% . Based on this 8-month period of monitoring, we could estimate the rate of ONL thinning to less than 3% per month. Of note, the inner segment/outer segment boundary was detected in C57BL/6J Nr2e3rd7/rd7 retinas on OCT at all time-points 16. The slow-progressing retinal degeneration observed in C57BL/6J Nr2e3rd7/rd7 mice resembles to what is observed in ESCS patients32. Because we detected increased expression of the non-apoptotic cell death markers PAR, Calpain-2 and Survivin, but not of the pro-apoptotic Bax, cleaved Caspase 3 and cleaved PARP-1 and the pro-necroptotic MLKL markers, our data suggest that the slow retinal degeneration observed in aging C57BL/6J Nr2e3rd7/rd7 retinas is driven by non-apoptotic cell death pathways, similar to what is observed in a vast majority of analyzed murine hereditary retinal degeneration models33.White spots and ‘rosettes’ gradually disappeared between 9 and 12 months of age in adult C57BL/6J 19. We observed an increased fluorescence signal in vivo in microglial cells around rosettes, which would be consistent with an increased uptake of hyperautofluorescent photoreceptor outer segments. Importantly, our analyses show an early phase of microglial cell migration into ‘rosettes’, followed by the immigration of monocytes/macrophages across the RPE  were kept in a 12-h light–dark cycle with unlimited access to food and water. In order to increase the fertility of the B6.Cg-Nr2e3rd7/J mice that had been backcrossed over 8 generations to homozygosity at Jackson Laboratory and had a typical litter size of 1–3 pups, we backcrossed them over 4 generations to the C57BL/6J genetic background, resulting in our C57BL/6J Nr2e3rd7/rd7 line with an average litter size of 6–8 pups. During backcrossing, all litters were systematically checked for the presence of the rd1 and rd8 mutations38. C57BL/6J Nr2e3rd7/rd7 mice were further crossed with homozygous mice selectively expressing green fluorescent protein (GFP) in microglia under the control of the Cx3cr1 gene were obtained by crossbreeding wild type Balb/cAnNCrl females with male transgenic homozygous fractalkine receptor reporter mice (Cx3cr1gfp/gfp) on a Balb/c background39.All experiments performed in this study were in accordance with the ARRIVE guidelines and were approved by the Veterinary Offices of the Canton of Bern (authorization BE17/19). C57BL/6J  and B6.Cg-® 23 gauge needle; Braun, Melsungen, Germany) of a ketamine 6 mg/ml  and medetomidine 0.1 mg/ml  solution, diluted in a sterile NaCl 0.9% solution, at 10 µl/g of body weight40. For mydriasis, eyes were rinsed with a drop of a sterile irrigating solution  for 10 s. Pupils were dilated with a subsequent drop of tropicamide 0.5%  for up to 2 min and of phenylephrine hydrochloride 100 mg/ml  for 1 min. After rinsing, the eyes were constantly humidified with 0.3% methyl-hydroxy-propyl-cellulose and dextran solution . The eye to be examined was then humidified with a drop of 2% hypromellose . Imaging was performed either on a retinal imaging microscope for small animals  or a confocal laser scanning ophthalmoscope . After funduscopy, mice were processed for OCT and/or fluorescein angiography. For OCT, retinal scans were acquired on both eyes of all animals and centered at the optic nerve head. Outer nuclear layer measurements were performed with InSight Software. For fluorescein angiography, anesthetized mice were injected intraperitoneally with a 5% sodium fluorescein solution  at a dose of 40 µl per 6 g of body weight. Anesthesia was reversed by intraperitoneal injection of an atipamezole 0.1 mg/ml solution , diluted in sterile NaCl 0.9%, at a concentration of 12 µl/g of body weight.Mice were anesthetized by intraperitoneal injection  and dried at room temperature for at least 1 h before being stained with hematoxylin–eosin 41. For immunohistochemistry, eyes were enucleated, fixed in 4% paraformaldehyde-1xPBS for 45 min at 4 °C, cryoprotected by immersion in 10% sucrose-1xPBS for 1 h, in 20% sucrose-1xPBS for 2 h and in 30% sucrose-1xPBS overnight at 4 °C, and 10-μM cryosections were collected as described above. Cryosections were hydrated with 1xPBS and blocked for 1 h in blocking solution . Then, all antibodies were diluted in blocking solution and incubated overnight at 4 °C: a rabbit polyclonal antibody directed against human S-opsin  was diluted 1/250, a rabbit polyclonal antibody against human L/M-opsin also cross reacting with mouse M-opsin  was diluted 1/1000, a rabbit polyclonal antibody against Iba1  was diluted 1/1000 and a rat monoclonal antibody against the macrophage F4/80 antigen  was diluted 1/250. Sections were then washed three times for 5 min in 1xPBS. Secondary antibodies conjugated to Alexa Fluor 488 or 594  were diluted at 1/1000 in blocking solution and incubated for 1 h at room temperature in the dark. Sections were washed 3 times for 5 min in 1xPBS and then counterstained for 10 min with 6-diamidino-2-phenylindole (DAPI) to visualize the cell nuclei. Slides were washed three times for 5 min in 1xPBS, before mounting in Citifluor . Images were acquired on a Leica MZ 16F stereomicroscope  or a Leica DM 6000B microscope. Confocal microscopy was performed on an inverted Zeiss LSM 710 fluorescence microscope  and Z-stacks of 100 µM with 5-µM intervals were acquired.For histochemical analysis, eyes were enucleated, fixed in 4% paraformaldehyde-1xPBS overnight at 4 °C, cryoprotected by immersion in 30% sucrose-1xPBS overnight at 4 °C and embedded in freezing compound . Ten-μm cryosections were collected on Superfrost® HardSet™ Antifade Mounting Medium containing DAPI . Rabbit polyclonal antibodies raised against cleaved PARP-1  and Calpain-2  were used at 1/100 in blocking solution and immunodetection was done with a secondary anti-rabbit Cy5 antibody  diluted 1/1000 in blocking solution. A mouse monoclonal antibody raised against PAR  was used at 1/100 in blocking solution and immunodetection was done with a secondary anti-mouse FITC antibody  diluted 1/1000 in blocking solution.For immune-detection of proteins in cell death mechanisms, 5 μM paraffin sections of retinas on SuperFrost Plus™ adhesion slides  were stained with rabbit polyclonal antibodies raised against Survivin , Bax , MLKL , and cleaved Caspase-3  diluted at 1/200 in blocking solution , 0.2% Triton X-100  in 1X PBS pH 7.4 . Immunodetection was done with a secondary anti-rabbit Alexa Fluor 594 antibody  diluted 1/1000 in blocking solution. Slides were mounted with VECTASHIELDhttp://imagej.nih.gov/ij; National Institutes of Health, Bethesda, Maryland USA). Measurements were done in dorsal and ventral regions on rectangles of 570 × 1370 pixels. Background was removed using in-built Otsu thresholding method with analyzed particle size (volume) set to 10–400 pixel2.Retinas were isolated under a dissecting microscope and gently transferred to a 96-well cell culture dish containing 4% paraformaldehyde-1xPBS for 2 h. They were washed three times for 5 min in 1xPBS and blocked for 1 h at room temperature with gentle agitation in blocking buffer . Incubation in primary antibody  was performed overnight at 4 °C with gentle agitation. Secondary antibodies, diluted 1/1000 in blocking solution, were incubated for 1 h at room temperature. Retinas were then washed 3 times in 1xPBS and transferred to a glass slide. To flatten the retinas, four cuts were made, equidistant apart, from the periphery of the retina towards the center. Slides were mounted in Citifluor before images were acquired on a Leica DM 6000B microscope. Cone outer segments were quantified using Fiji/ImageJ version 1.51 (TMPlus).On ice, two mouse retinas were homogenized with a plastic pestle in 100 µl of a buffer containing 100 mM NaCl, 50 mM Tris pH 7.5, 1 mM EDTA, 0.1% Triton X-100, and freshly added protein inhibitors . Thirty micrograms of protein extracts were resolved on 10% SDS-PAGE gels followed by transfer on PVDF membrane . Membranes were blocked in 5% non-fat dried milk before being immunoassayed using rabbit polyclonal antibodies against survivin , S-opsin and M-opsin  and a mouse monoclonal antibody against α-tubulin . The secondary ECL™ donkey anti-rabbit and sheep anti-mouse IgG horseradish peroxidase-conjugated antibodies were diluted 1/15,000 . Proteins were detected by chemiluminescence using the Amersham ECL Advance Western Blotting Detection Kit  in a Fujifilm LAS-4000 mini imaging system . Molecular weight markers were purchased at Fermentas .Supplementary Information.


Phanerochaete chrysosporium (PcBxl3), and the other from the ascomycete Trichoderma reesei (TrXyl3A). A comparison of the crystal structures of the two enzymes, both with saccharide bound at the catalytic center, provided insight into the basis of substrate binding at each subsite. PcBxl3 has a substrate-binding pocket at subsite -1, while TrXyl3A has an extra loop that contains additional binding subsites. Furthermore, kinetic experiments revealed that PcBxl3 degraded xylooligosaccharides faster than TrXyl3A, while the KM values of TrXyl3A were lower than those of PcBxl3. The relationship between substrate specificity and degree of polymerization of substrates suggested that PcBxl3 preferentially degrades xylobiose (X2), while TrXyl3A degrades longer xylooligosaccharides. Moreover, docking simulation supported the existence of extended positive subsites of TrXyl3A in the extra loop located at the N-terminus of the protein. Finally, phylogenetic analysis suggests that wood-decaying basidiomycetes use Bxls such as PcBxl3 that act efficiently on xylan structures from woody plants, whereas molds use instead Bxls that efficiently degrade xylan from grass. Our results provide added insights into fungal efficient xylan degradation systems.Xylan is the most common hemicellulose in plant cell walls, though the structure of xylan polymers differs between plant species. Here, to gain a better understanding of fungal xylan degradation systems, which can enhance enzymatic saccharification of plant cell walls in industrial processes, we conducted a comparative study of two glycoside hydrolase family 3 (GH3) β-xylosidases (Bxls), one from the basidiomycete  Woody and herbaceous biomasses are sustainable sources of biofuel and bio-based chemicals that can replace fossil resources, and enzymatic saccharification is a mild and effective way to utilize them. In nature, fungi degrade polysaccharides such as cellulose and hemicellulose, which account for about 70% of plant cell walls, with producing a variety of enzymes to support their growth. Therefore, the characterization of these enzymes can provide an insight into not only fungal degradation system, but also efficient enzymatic saccharification of the cellulosic biomass. Since some hemicelluloses are present on cellulose crystals, degradation of hemicellulose is crucial. Xylan is a common hemicellulose in the secondary cell wall of Angiosperms . The maiBxls are key enzymes for fungi to utilize xylan, because, unlike xylanase, they efficiently produce the monosaccharide xylose. Although Bxl works at the end of the xylan degradation system, it has to degrade a variety of substrates because xylanases produce diverse types of small xylooligosaccharides with various DPs and side chains, depending on the type of target polysaccharide .http://www.cazy.org/) , the PA14 domain supports binding of longer substrates (Glycoside hydrolases (GH) family 3 (GH3) is one of the largest families in the Carbohydrate-Active enZymes (CAZy) database zy.org/)  and inclbstrates . In the bstrates . But, inHypocrea jecorina   and 23-α-l-arabinofuranosyl-xylotetraose (XA2XX)  , TrXyl3Acharides . T. rees biomass . For ins, and 30 , 13. How (XA2XX) . In contot fungi . It prodot fungi , as wellP. chrysosporium and T. reesei. The ligand-bound structures of these two enzymes account well for the kinetic differences, which are consistent with the evolution of the different xylan-degrading machineries required to degrade the preferred growth substrates of basidiomycetes and ascomycetes.Here, we describe structural and kinetic comparisons of two β-xylosidases from PcBxl3 and TrXyl3A contain 743 and 777 amino acid residues, respectively. Since the sequence identity between the two enzymes is 54%, the distributions of secondary structures in the two GH3 enzymes are quite similar, as shown in TrXyl3A. This additional N-terminal region containing the α-helix of TrXyl3A consists of 27 amino acids. There are also ten extra amino acids at the C-terminal region of TrXyl3A, but no secondary structure is predicted for them.PcBxl3 and TrXyl3A, the crystal structures of both enzymes were solved. The crystal structure of TrXyl3A bound with thioxylobiose was determined by molecular replacement (MR) using free TrXyl3A (Protein Data Bank (PDB) ID: 5A7M, chain B) as a search model, and the crystal structure of free PcBxl3 was determined by MR using TrXyl3A (5AE6 chain B). The structure of PcBxl3 with xylose bound in the catalytic center was also determined. The data collection statistics and structural refinement statistics are summarized in To uncover the structural differences between PcBxl3 consists of three domains: (β/α)8 domain, (α/β)6-sandwich domain, and fibronectin III (FnIII)-like domain. The overall structure and order of these domains  suggests that PcBxl3 has Glu470 as a possible acid/base amino acid and Asp267 as a catalytic nucleophile, while the corresponding residues in TrXyl3A are Glu492 and Asp291. The conformation of Glu470 (PcBxl3) is the same in the native and liganded structures of PcBxl3, but Glu492 (TrXyl3A) has different conformations in native and liganded TrXyl3A.The catalytic residues of GH3 enzymes are glutamic acid and aspartate , 18, 19.B, both Bxls have a pocket at subsite -1. The binding mode of a xylose residue at subsite -1 is important for substrate recognition and to maintain the substrate in a suitable conformation during the catalytic reaction. There are three important interactions, as illustrated in C and D: the interactions with the C2, C3, and C4 hydroxyl groups , the interaction with C5, and the interactions with the α- and β-faces of the xylose ring, which are named according to the usual rules  of the xylose residue bound at subsite −1 are also similar in the two enzymes. Glu62 and Lys183 are located at distances of 2.7 and 3.0 Å from O4 of the xylose residue in PcBxl3, respectively, and Glu89 and Lys206 in TrXyl3 are both located at a distance of 2.7 Å from O4. In addition, Trp60 of PcBxl3 and Trp87 of TrXyl3A are located near C5. Thus, tryptophan interacts with C5 hydrophobically. The environments opposite the α- and β-faces of the xylose ring are significantly different. The hydroxyl group of Tyr407 of PcBxl3 and that of Tyr429 of TrXyl3A are oriented toward the α-face, while a disulfide bond and methionine are exposed to the β-face. The amino acids surrounding the nonreducing end sugar moiety are well conserved, but PcBxl3 has Phe129 as the residue corresponding to Tyr152 of TrXyl3A, indicating that subsite −1 of PcBxl3 would be more hydrophobic than that of TrXyl3A.As shown in Figure 3al rules , as showPcBxl3, there is no clearly detectable subsite +1 , but not 13-acetyl xylobiose (XA3) (HvExoI (1IEX)  (In bsite +1 . In contse (XA3) . CompareI (1IEX)  and Bgl A, 4I8D) , subsite2), xylotriose (X3), xylotetraose (X4), xylopentaose (X5). DPs of them are thought to be those of major products of xylan degradation by xylanases. As expected from the structural differences between PcBxl3 and TrXyl3A, the kinetic parameters are significant different n+1/(kcat/KM)n reflects the binding affinity at subsite n. Based on the maximum value of kcat, kint of PcBxl3 and TrXyl3A was assumed to be 2.05 and 0.82 s−1, respectively. The results are shown in PcBxl3. On the assumption of two binding modes, the subsite affinity at subsite −1 can be derived from kcat and the affinities of subsites +2 to +4  has a higher affinity than the sum of the affinities of other subsites. TrXyl3A shows higher affinity at the active center than PcBxl3, and the difference is about 1 kcal/mol, which is consistent with the structural findings.The subsite affinities  of both +2 to +4 . HoweverTrXyl3A has a binding energy of 1 kcal/mol, which is typical for subsites of other GH , and X3 (TrXyl3A) were estimated by docking simulation; the results are summarized in PcBxl3 and as that of thioxylobiose in the crystal structure of TrXyl3A, supporting the reliability of the simulation results. In the case of PcBxl3, X2 showed the highest score, −6.3 kcal/mol. In the case of TrXyl3A, X2 and X3 showed the highest score, −7.9 and −8.9 kcal/mol, respectively. The difference between the scores of the X2 and X3 binding modes is -1.0 kcal/mol, which is consistent with the subsite affinity in 2 binding modes is -1.7 kcal/mol.Next, the affinities of xylose and X2 binding modes of PcBxl3 and TrXyl3A, the score of PcBxl3 is about a half of that of TrXyl3A. As shown in Figure 6B, only O3 of the xylose residue at subsite +1 can have a hydrophilic interaction with Tyr407 (PcBxl3) and Tyr429 (TrXyl3A). Further, as shown in Figure 6C, less than four atoms of the xylose residue at subsite +1 interact with PcBxl3, whereas six atoms of xylose with the residue at subsite +1 interact with TrXyl3A. Thus, the difference between the scores is thought to be mainly due to the difference in number of hydrophobic interactions at subsite +1.Comparing the scores of the xylose and XTrXyl3A docked with X3 in Figure 6B, Leu17 is located at a distance of 3.0 Å from C5 of the xylose moiety at subsite +2. Additionally, two water molecules on the surface of the extra loop are placed near the xylose moiety, at a distance of 2.9 Å. They may have a role in binding the substrate, because the results of docking simulation without water gave a significantly lower score (data not shown). These results support that the idea TrXyl3A contains subsite +2 and suggest that the extra loop is important for binding longer oligosaccharides.Focusing on PcBxl3 (7VC7), TrXyl3A (5AE6), TrCel3A (4I8D) (HvExoI (1IEX)   and HvExI (1IEX) . Most off PcBxl3 , this tyPcBxl3 and TrXyl3A are significantly different. In the crystal structure of PcBxl3, subsite +1 was not clearly identified, but that of TrXyl3A was clearly visualized in the crystal structure. The isoprimeverose (α-d-xylopyranose-(1→6)-d-glucopyranose derived from xyloglucan)-producing enzyme belonging to GH3 from Aspergillus oryzae has a shallow pocket adjacent to subsite −1, broadening it to subsite −1’ . Thus, any Bxl in this environment should preferentially degrade small xylooligosaccharides. Brown-rot fungi also have GH10 xylanases, but favor softwood substrates, which include arabinoglucuronoxylan. The structure of arabinoglucuronoxylan is simpler than that of acetyl glucuronoxylan in that glucuronic acid substitution mostly occurs once every six xylose residues, and arabinose is located two residues away from a GlcA-substituted residue (4 derived from XUXX and 13-α-l-arabinofuranosyl-xylobiose (XAXX). GH30 xylanases produce longer xylooligosaccharides depending on the substitution patterns. Furthermore, some substituted xylooligosaccharides need to be digested by Bxl before debranching  (PcBxl3 was obtained from Joint Genome Institute (JGI) MycoCosm (Protein ID: 2919526) (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins) was used for calculation of identity. The amino acid sequences were aligned using Clustal W (TrXyl3A (Amino acid sequence of nih.gov) . Amino a2919526) . The Blalustal W . Seconda. The cloned fragment was amplified with the gene-specific primers, Pcxyn3_9257_Met (5′- ATGGTCGCCAGCCCAGATTTAAGC-3′) and Pcxyn3_9257_Met (5′- GCTGCCTATGTACACTACCGTGGTTG -3′), and ligated into pPICZα A plasmid vector (Invitrogen) using EcoRI and NotI restriction recognition sites. After transformation of Pichia pastoris strain KM71H by electroporation, transformants were selected with Zeocin according to previous research , subjected to 100 kDa ultrafiltration, and concentrated using a 5 kDa filter (Merck Millipore).The recombinant proteins were produced using a 5 L jar fermenter  with methanol feed according to previous reports , 46, 47.PcBxl3 and TrXyl3A, Phenyl Toyopearl 650M  was used for the first chromatography. After equilibration with 50 mM sodium acetate buffer (pH 5.0) containing 1 M ammonium sulfate (Wako), the enzymes were eluted with 50 mM sodium acetate buffer (pH 5.0). TSKgel DEAE-5PW  was used for further purification. After equilibration with 50 mM Tris-HCl buffer (pH 8.0), the enzymes were eluted with 50 mM Tris-HCl buffer (pH 8.0) containing 1 M NaCl (Wako). The concentration of the purified proteins was quantified by NanoDrop2000 (Thermo Fisher Scientific)The resulting solutions were purified by two-step column chromatography. For the characterization of PcBxl3, deglycosylation was conducted by endoglycosidase H from Streptomyces plicatus before the DEAE column chromatography. The pET 28 vector with the gene encoding endoglycosidase H was a generous gift from Professor Satoshi Kaneko. Transformation of Escherichia coli BL21(DE3) (New England Biolabs) was conducted according to the manufacturer’s instructions. After cultivation in LB medium, protein expression was induced with 100 μM IPTG at 37 °C. The medium containing cells was centrifuged at 10,000g for 10 min, and cell lysis was performed using BugBuster (Merck) according to the manufacturer’s instructions. The recombinant endoglycosidase H was purified using HisTrap HP column (GE). After equilibration with 20 mM Tris-HCl buffer (pH 7.0) containing 500 mM imidazole (Wako), the enzyme was eluted with 20 mM Tris-HCl buffer (pH 7.0). The protein solution of PcBxl3, without denaturing, was mixed with 0.2 mg purified endoglycosidase H per 1 mg of PcBxl3 and sodium acetate buffer (pH 6.0). The mixed solution was incubated at 37 °C for 24 h.For crystallization of TrXyl3A was a kind gift from DuPont Industrial Bioscience. The protein was stored prior to crystallization experiments at 4 °C in a stock solution containing 149 mg/ml protein, 13% sorbitol, and 0.125% sodium benzoate in 0.1 M Sodium acetate (pH 5.0).The protein used for crystallization of PcBxl3 was conducted by sitting-drop vapor diffusion method using a 96-well sitting drop plate (Greiner). For crystallization, 0.5 μl of 10 mg/ml PcBxl3 was mixed with 0.5 μl of the reservoir solution containing 0.2 M lithium sulfate, 0.1 M Tris (pH 8.5) and 40 v/v % PEG 400 . The plate was incubated at 293 K for 3 weeks. The reservoir solution for soaking contained 525 mM malic acid  and 20 v/v % PEG 3350 (Sigma-Aldrich). The mixture was incubated at 293 K for 1 week. The crystal was soaked in 40 v/v % PEG 400 (Sigma-Aldrich) and 100 mM X2 (Megazyme) for a short time.The crystallization of TrXyl3A protein stock solution was diluted to 10 mg/ml by adding 0.1 M sodium acetate buffer (pH 4.5) just prior to crystallization. Optimized TrXyl3A crystals for data collection were obtained by the hanging drop vapor diffusion method. Crystals for ligand-bound TrXyl3A data collection were obtained using the PACT screen (Qiagen) condition C4 (0.1 M PCB (pH 7.0) and 25% PEG 1500). 4-thioxyloboise was chemically synthesized according to the previous research  and 33% PEG 1500, with 14 mM 4-thioxylobiose. Prior to data collection, the TrXyl3A crystals were briefly incubated in a cryoprotectant solution containing 30% PEG 3350 and 10% glycerol and then flash-frozen in liquid nitrogen.The research . SoakingTrXyl3A crystal soaked with 4-thioxylobiose (to 2.1 Å resolution) was collected at beamline I911-5, at MAXII-lab, Sweden. All TrXyl3A diffraction data were processed using the data integration program Mosflm (TrXyl3A 4-thioxylobiose ligand structure was solved by molecular replacement (MR) using the program Phaser  as search model.The dataset of a m Mosflm  and scalm Mosflm . The TrXm Phaser , 53 usinTrXyl3A ligand-bound structure was performed using the program REFMAC5 , High Energy Accelerator Research Organization. Diffraction data were collected on a DECTRIS PILATUS3 S 6M (Dectris). Crystals were cryocooled in a nitrogen gas stream to 95 K. The crystal data of free PcBxl3 were integrated and scaled using XDS installed in PREMO. The data for the crystal soaked in X2 solution were integrated and scaled using XDS and Aimless in the CCP4i2 program suite. Structural determination of PcBxl3 was conducted by molecular replacement with MOLREP in the CCP4i2 program suite. N-Acetyl-d-glucosamine, xylose, water molecules, and crystallization agents were modeled based on the electron density map and the coordination distances. The orientation of xylose molecule was determined by mimicking that of 5A7M at subsite −1. Refinement was conducted by Refmac5 in CCP4i2  (TrXyl3A), and 7VC6 and 7VC7 (PcBxl3), respectively.The coordinates for the final nk (PDB) , 61, 62,PyMOL  was used for structural drawings. Ligand–protein interaction was depicted using Ligplot+ (v.2.2) 2), xylotriose (X3), xylotetraose (X4), and xylopentaose (X5) (Megazyme) were used as substrates. The reaction mixture consisted of enzyme, 5 mM sodium acetate buffer (pH 5.0), and various concentrations of xylooligosaccharides in the range from 50 μM to 5 mM. The concentrations of PcBxl3 and TrXyl3A were 30.2 nM and 26.6 nM, respectively. The reaction was performed at 30 °C for 30 min and stopped by heating the mixture at 95 °C for 5 min to prevent the reaction from continuing during quantification. Measurement of the amount of released xylose was performed by high-performance liquid chromatography  with tandem columns of SUGAR-KS802 and 801 (Showa Denko). The samples were manually injected. The column oven was set at 70 °C. A Corona CAD detector (ESA Biosciences) was used.Xylobiose   in Chime2 server , 67.TrXyl3A bound with thioxylobiose), 7VC6 (native form of PcBxl3), and 7VC7 .The structures presented in this paper have all been deposited in the Protein Data Bank (PDB) with the following codes: 5AE6 (crystal structure of This article contains The authors declare that there is no conflict of interests with the contents of this article.


The resulting BBB displayed continuous tight-junction patterns, low permeability to mannitol and sucrose, and quasi-physiological responses to hyperosmolar opening and p-glycoprotein inhibitor treatment, as demonstrated by decreased BBB integrity and increased permeability of rhodamine 123, respectively. Astrocytes and pericytes on the abluminal side of the vascular channel provided the environmental cues necessary to form a tight barrier and extend the model’s long-term viability for time-course studies. In conclusion, our novel multi-culture microfluidic platform showcased the ability to replicate a quasi-physiological brain microvascular, thus enabling the development of a highly predictive and translationally relevant BBB model.Microfluidics-based organ-on-a-chip technology allows for developing a new class of in-vitro blood-brain barrier (BBB) models that recapitulate many hemodynamic and architectural features of the brain microvasculature not attainable with conventional two-dimensional platforms. Herein, we describe and validate a novel microfluidic BBB model that closely mimics the one in situ. Induced pluripotent stem cell (iPSC)-derived brain microvascular endothelial cells (BMECs) were juxtaposed with primary human pericytes and astrocytes in a co-culture to enable BBB-specific characteristics, such as low paracellular permeability, efflux activity, and osmotic responses. The permeability coefficients of [ Central nervous system (CNS) disorders have limited treatment options, although they are significant causes of morbidity and mortality worldwide . A consiBBB impairment is implicated in the pathogenesis and progression of various neurological diseases ,8,9,10. Microfluidics-based organ-on-a-chip technology has led to the recent development of a new class of in vitro models ,16. Thes13C12] sucrose and [13C6] mannitol. We co-cultured iBMECs with primary human pericytes and astrocytes on the organ-on-a-chip model to emulate BBB-specific characteristics, such as high barrier function, low permeability, and efflux activity. We also showed that the organ-on-a-chip model could maintain the barrier function for 1 week, whereas traditional iBMECs Transwell models can maintain the barrier function for only 2 days.This study validated the barrier function of a BBB-on-a-chip platform using  sucrose and [13C6] mannitol. For this purpose, 100 μg/mL of [13C6] mannitol and [13C12] sucrose  was added simultaneously to the vascular (lumen) channel. Samples were collected over specific time intervals from the apical channel (ablumen) and measured using an established UPLC-MS/MS method [2H8] mannitol and [2H2] sucrose  as an internal standard followed by centrifugation at 12,000 rpm for 10 min. The supernatant was transferred into autosampler inserts and then injected into the LC-MS/MS. An Acquity BEH amide column  was used for chromatographic separation using acetonitrile:water:ammonium hydroxide  as the mobile phase, at a flow rate of 0.2 mL/min. Electrospray ionization in negative mode was used as the ionization source, and data acquisition and quantification were made using Analyst software.Barrier integrity of a BBB microfluidic chip was obtained by measuring the permeability coefficients of stable isotopes of  Sucrose (100 µg/mL) was simultaneously added to monitor the barrier integrity. Effluents were collected from the apical and basal channels and quantified using fluorescence intensity. The fluorescence was measured at 485/530 nm to quantify the rhodamine 123 concentration via a SynergyMX2 ELISA plate reader . The BBB permeability of rhodamine 123 and sucrose was measured in the presence or absence of the inhibitor.The P-gp functionality was evaluated using rhodamine 123 (Sigma-Aldrich), a substrate of the P-gp transporter ,29. Both13C12] sucrose and [13C6] mannitol. The permeability coefficients of the markers were obtained at different time points to evaluate the BBB opening and recovery.A hypertonic solution containing 25% mannitol was introduced into the vascular channel at a 120 μL/h flow rate for 10 min. Mannitol was then replaced with 100 μg/mL of  sucrose and [13C6] mannitol, known as one of the ideal paracellular permeability markers [2) to investigate the difference in barrier properties. Results show that the permeability of [13C12] sucrose and [13C6] mannitol across the single culture of the iBMEC monolayer was 1.883 × 10−6 ± 3.763 × 10−7 and 2.380 × 10−6 ± 5.738 × 10−7 cm/s , respectively, after 2 days in the culture, while the permeability of sucrose and mannitol in the triculture was 4.923 × 10−7 ± 1.187 × 10−7 and 6.760 × 10−7 ± 1.071 × 10−7 cm/s , respectively . Accordingly, our results strongly suggest that a co-culture with primary human astrocytes and pericytes can enhance the barrier function of the BBB on a chip. Furthermore, permeability measurements were performed at 2, 5, 7, and 10 days post-setup , respectively, whereas it was 1.166 × 10−6 ± 3.277 × 10−7 and 2.180 × 10−6 ± 3.306 × 10−7 cm/s on day 7 and day 10 , respectively. The permeability on days 2 and 5 was significantly different from that measured on day 10 . A similar trend was observed when measuring mannitol permeability (n = 3), which increased from 6.640 × 10−7 ± 3.651 × 10−8 and 6.430 × 10−7 ± 3.557 × 10−8 on day 2 and day 5 to 3.053 × 10−6 ± 5.258 × 10−7 on day 10 .We assessed the passage of  sucrose and [13C6] mannitol to assess the impact of the hyperosmolar challenge on BBB integrity as a function of permeability. −7 ± 1.22 × 10−7 cm/s pre-opening to 6.910 × 10−6 ± 2.014 × 10−6 cm/s 1 h following the hyperosmolar challenge. A similar trend was observed for mannitol permeability (increasing from 8.253 × 10−7 ± 1.436 × 10−7 cm/s pre-opening to 7.983 × 10−6 ± 2.398 × 10−6 cm/s 1 h post-opening). A longitudinal assessment of paracellular permeabilities showed that the BBB integrity started recovering at 2.5 h after the hyperosmolar challenge  and was fully restored at 24 h .We infused a hyperosmolar mannitol solution into the vascular channel for 10 min. We then immediately perfused the lumen with [13C12] sucrose and [13C6] mannitol permeability values in a microfluidic chip were 4.923 × 10−7 ± 1.187 × 10−7 and 6.760 × 10−7 ± 1.071 × 10−7 cm/s  to assess the integrity and tightness of the BBB ,18,19. Hcm/s see A,B, resp−7 cm/s. For instance, the permeability coefficient of 10 kDa dextran was shown to be 3.1 × 10−7 cm/s in rat cerebral microvessels. A drawback of such imaging techniques is the lack of quantitative uptake measurement and vessel damage resulting from the grounding of the skull. As a result, these techniques could lead to the overestimation of dextran permeability levels. Studies, for example, by Mehvar et al., have demonstrated that even with 4 kDa dextran, there is no fluorescein-labeled dextran concentration in brain tissue  sucrose permeability, which closely mimics previous results obtained by our group in vivo using a mouse model undergoing transient BBB opening by hyperosmolar mannitol  sucrose and [13C6] mannitol in the microfluidic model. The BBB microfluidic model displays critical tight-junction proteins, an efflux pump, and transporters. Moreover, the physiological barriers were maintained until day 7, which could be used for any chronic disease studies. We also demonstrated successful modulation of both transcellular and paracellular permeability using the P-gp inhibitor and hyperosmolar agent mannitol. Our results suggest that the multi-culture microfluidic model can effectively reproduce a quasi-physiological microenvironment, allowing for developing a highly predictive and translationally relevant BBB model. This novel platform can be a valuable tool for screening putative CNS-targeting drugs and assess the feasibility and effectiveness of novel delivery methods across the BBB.This study validated the barrier function of a human iPSC-derived blood-brain barrier microfluidic chip by using small-molecular-weight markers. Sucrose and mannitol are known as ideal standard markers for the measurement of BBB integrity in in vivo studies. We showed low permeability coefficient values for [