Commonly, the SE-involved genes are identified by analyzing the heterogeneous population of explant cells and thus, it is necessary to validate the expression of the candidate genes in the cells that are competent for embryogenic transition. Here, we optimized and implemented the whole mount ) method  in order Because SE results in the efficient production of somatic embryo-derived regenerates, it has become a powerful tool in plant biotechnology, and is widely applied in the clonal propagation and genetic transformation of numerous plant species , which is required for miRNA biogenesis, have been found to inhibit SE-induction in vitro in cultures of Arabidopsis explants  involves the formation of somatic embryos in somatic cells that are cultured reviewed . Moreovereviewed . Variousreviewed , the cenexplants . The hypnduction . Similara sativa , hybrid a sativa , Larix lptolerix , Dimocars longan , Gossypihirsutum , and ZeaZea mays .in situ hybridization (WISH) protocol for miRNA and mRNA molecules in explants that are undergoing SE induction.The current limitation for identifying SE-enriched genes/miRNA is to distinguish the cells that are triggered toward embryogenic development from the surrounding explant cells. One way to examine whether the expression of the analyzed gene is present in cells that are undergoing SE is to analyze the reporter lines, which are based on the GUS or GFP constructs. However, the overwhelming majority of the available reporter lines monitor promoter activity rather than lead to the localization of the corresponding transcript. In order to overcome the time-consuming and laborious processes that are required to prepare a specific construct and plant transformation, we present the whole mount Arabidopsis thaliana (L.) Heynh. Columbia (Col-0) parental genotype and the transgenic lines 35S::MIR156 (N9952) and 35S::MIM156 (N9953) were supplied by the Nottingham Arabidopsis Stock Centre (NASC). The seeds of the 2x35S::STTM165/166 line (hereafter referred to as STTM165/166) were kindly provided by Dr. Guiliang Tang , while the seeds of the MIR167a::GUSP and MIR167c::GUSP lines were kindly provided by Jason W. Reed from the Department of Biology, University of North Carolina at Chapel Hill.The -2s-1 white, fluorescent light. The young seedlings were transferred into Jiffy-7 pots and grown in a walk-in type green room under the conditions that are described above until the siliques were harvested.The seeds were sterilized using a 20% solution of commercial bleach and then plated on a solid 1/2 MS medium. The plates were kept in a growth chamber at 21 ± 1°C under a 16/8 h photoperiod of 40 μM mImmature zygotic embryos (IZEs) at the green cotyledonary stage were used as the explants to induce SE. The IZEs were cultured in an E5 solid medium with 5 μM 2,4-dichlorophenoxyacetic acid  according to Explants from different time points of the SE culture were collected and fixed in 4% PFA in PBS with 0.1% Tween in a vacuum for 3 h at room temperature (RT). Next, the samples were rinsed for 30 min in methanol and then washed 3 × 5 min in 100% ethanol. After fixation, the samples were washed in PBS and dehydrated in an ethanol series  for 10 min each, followed by replacing the ethanol with acetone. The dehydrated samples were dried with a CPD 2 critical-point drier (Pelco) using liquid carbon dioxide, mounted on aluminum stubs with double-sided adhesive carbon tape and sputter-coated with a 12.5 nm  or 20 nm (21 and 28 days) film of gold in an SC-6 sputter coater (Pelco). After processing, the samples were imaged using a Hitachi SU 8010 UHR FESEM field emission scanning electron microscope  at a 5 kV accelerating voltage with a secondary electron detector (ESD).After stopping the colorimetric detection, the explants were transferred to three-welled glass slides, mounted in 70% glycerol in a TE buffer or Clear Solution and sealed with cover-slips. The slides were subsequently imaged on an automated Pannoramic SCAN 150 slide scanner (3D HISTECH) with transmitted light and a 20X plan-apochromat objective or with an Olympus BX-43 microscope. Images of the embryos were collected using the Panoramic Viewer software (3DHISTECH) or with an Olympus SC-30 camera using the CellSens software.For the miRNA WISH procedure, LNA-modified oligonucleotide probes that corresponded to the full antisense sequence of the miRNA were DIG-labeled at both the 5′ and 3′ ends that contained 4 LNA-modified bases at positions 7, 9, 11, 15. The probes were synthesized using Exiqon.in vitro transcription reaction using an NTP Labeling Mix.The probes that were used for the mRNA WISH were synthesized as ∼450 bp cDNA transcripts, and then cloned into pSPT19. The T7/SP6 RNA polymerases were used to generate DIG-labeled probes by an • Plant material: immature zygotic embryos that were cultured on an SE induction medium for 0, 5, 10, 15 days); 60 explants per culture combination• Formamide • NBT/BCIP Stock solution • Anti-Digoxigenin-AP • 1-Methylimidazole • N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride • BSA solution 30% • Denhardt’s solution • Dextran sulfate 50% solution • tRNA 50 mg/ml • Histo-Clear II • SSC buffer 20 × concentrate • Glycine • Tween-20 • Proteinase K • Sodium acetate • T7 RNA polymerase • SP6 RNA polymerase • NTP Labeling Mix • Methanol • Ethanol • Formaldehyde solution 37% • RNaseZAP • Medium incubation baskets (100 μm) • 24-well cell culture clusters • “PTFE” Slides • ExPellPlus Filter Tip • Glass bottles, sterile, nucleases free (baked 8h at 180°C)• RNase-free Microfuge Tubes (1.5 mL) • Cover slips 24 mm × 50 mm (Menzel-Glaser)• Tweezers•2O; add pure HCl till pH 7.5; 8.0 or 9.0 Tris–HCl 1 M [250 ml]: 30.275 g Tris + up to 250 ml H•2O; add crystals of NaOH (∼0.5 g) till pH 8 EDTA 0.5 M [250 ml]: 46.525 g EDTA + up to 250 ml H•2O NaCl 5 M [250 ml]: 73.05 g NaCl + up to 250 ml H•2 1 M [100 ml]: 20.3 g MgCl2 × 6H2O + up to 100 ml H2O MgCl•2O; pH 7.5 RNase buffer 5× [500 ml]: 25 ml 1 M Tris–HCl pH 7.5 [50 mM] + 5 ml 0.5 M EDTA [5 mM] + 250 ml 5 M NaCl  + up to 500 ml H•2O Clearing solution [1 ml]: 2.5 g chloral hydrate in 1 ml 30% glycerol in H•2CO3 [120 mM] + 0.672 g NaHCO3 [80 mM] + up to 100 ml H20 – DEPC; pH 10.2 [NaOH] 2× carbonate buffer [100 ml]: 1.277 g Na•2PO4 + 25.07 g Na2HPO4+ up to 1000 ml H2O; pH 7.0 [HCl] PBS 10× [1000 ml]: 76 g NaCl + 4.14 g NaH• Proteinase K buffer [200 ml]: 100 ml 1 M Tris pH 7.5 + 100 ml 0.5 M EDTA + 0.5 ml acetic anhydride (DO NOT TREAT WITH DEPC!)• PBST: PBS + 0.1% Tween (1 μl of Tween per 1 ml PBS)•2HPO4 1 M [1000 ml]: 141.96 g Na2HPO4 + up to 1000 ml H2O Na•2PO4 1 M [1000 ml]: 138 g NaH2PO4 + up to 1000 ml H2O NaH•2HPO4 1 M + 53.7 ml NaH2PO4 1 M; pH 6.8 Na-phosphate [100 ml]: 46.3 ml Na•In situ salts 10× [10 ml]: 6 ml 5 M NaCl + 1 ml 1 M Tris pH 8.0 + 1 ml 1 M Na-phosphate + 1 ml 0.5 M EDTA + 1 ml H20•in situ salts + 0.2 ml 50× Denhardt’s salts + 0.1 ml tRNA 50 mg/ml + 1.7 ml H2O-DEPC (fresh) Hybridization Mix [10 ml]: 5 ml formamide + 2 ml 50% dextran sulfate + 1 ml 10× •2 + 830 ml H2O-DEPC (fresh) TNM-50 [1000 ml]: 100 ml 1 M Tris pH 9.5 + 20 ml 5 M NaCl + 50 ml 1 M MgCl•2O TE 10× [1000 ml]: 100 ml 1 M Tris pH 8.0 + 20 ml 0.5 M EDTA + up to 1000 ml H•2O; pH 8.0/HCl (pH is very important!) Methylimidazole-NaCl [1000 ml]: 10.36 ml methylimidazole + 75 ml NaCl 5 M + up to 1000 ml H• EDC [1000 ml]: 31.632 g N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride + up to 1000 ml methylimidazole-NaCl pH 8.0The available protocol of WISH  was modi1.® 20) on ice. The tissue was fixed under a vacuum for 3 h at RT. Depending on the age of the explants in the culture, different approaches were developed. Freshly isolated explants from the siliques (0 day of culture) were dissected in a drop of PBS, while the explants after 5, 10, and 15 days of SE culture were transferred directly from the medium onto 3.7% PFA in PBST , then twice in 100% ethanol for 5 min each at RT.! NOTE: For 10 days and older tissues, the incubation in methanol can take up to 1 h at RT.⊕ PAUSE: The fixed and dehydrated tissue can be stored in 100% ethanol at -20°C for several weeks.3. Incubate for 30 min in 1:1 Histo-Clear II and a 100% ethanol solution at RT.4. Wash twice for 5 min in 100% ethanol at RT.5.2O, 10 min each at RT. Rehydrate in 90, 70, 50, and 30% ethanol/H6. Incubate in a proteinase K buffer for 5 min at RT.7. Incubate with proteinase K (75 μg/ml) in a proteinase K buffer for 15 min at 37°C.! NOTE: The proteinase K buffer should be preheated to 37°C.8. Stop the digestion using glycine (2 mg/ml) in PBS by incubating it for 5 min at RT.9. Wash for 5 min in PBST at RT.10. Refix in 3.7% PFA in PBST for 10 min at RT.11. Wash twice in PBST for 5 min each at RT.12. Incubate three times for 10 min each in methylimidazole-NaCl (pH 8.0) at RT.13. Incubate for 2 h in 0.16 M EDC in methylimidazole-NaCl at 60°C.14. Wash twice for 10 min in PBST at RT.15. Wash in a pre-hybridization mix (hybridization mix without the probe) for 10 min at RT.16. Denature the probe in the hybridization mix for 2 min at 80°C.17. Incubate 12 h or overnight at 65°C in the hybridization mix with the denatured probe.! NOTE: The hybridization mix should be prepared fresh shortly before use.! NOTE: The best probe concentration was 20 nM.! NOTE: The incubation at 65°C may be carried out in a water bath in a 24-well plate that is sealed by Parafilm or in a humidified chamber.18. Wash twice for 10 min in 2×SSC at RT.19. Incubate twice for 30 min in 0.2×SSC at 65°C.! NOTE: During the incubation in 0.2×SSC at 65°C, the probes should be shaken from time to time OR the incubation can be carried out on a shaker at 65°C.20. Wash 5 min in PBST at RT.21. Incubate in 1% BSA in PBST for 30 min on a shaker at RT.22. Incubate with Anti-Digoxigenin-AP (1:1250) in 1% BSA in PBST for 90 min on a shaker at RT.23. Incubate in 1% BSA in PBST for 30 min on a shaker at RT.24. Wash twice for 10 min in PBST at RT.25. Wash three times in TNM-50, 5 min each at RT.26. Incubate in NBT-BCIP (1:50) in TNM-50 at RT.! NOTE: The TNM-50 buffer and NBT-BCIP solution should be freshly prepared, shortly before use.! NOTE: The incubation in NBT-BCIP can be carried out at RT or at 37°C in the dark. If the incubation is at 37°C, the colorimetric reaction should be checked every 10 min.! NOTE: During the incubation at RT colorimetric reaction for miR390 takes 1 h, while takes at least 3 h for miR124 and miR166.27. Stop the reaction using TE and wash three times for 5 min in TE at RT.28. Carefully transfer the explants in TE onto glass slides.29. Remove the TE and add the 70% glycerol in TE or clearing solution, gently cover with a cover slip.30. Perform the microscope analysis.! NOTE: Do not allow the explants to remain in the clearing solution for longer than 30 min.To perform the mRNA WISH in explants that are undergoing the SE process, we modified and adjusted the procedure based on alkaline phosphatase (AP) coupled antibody detecting the anti-Digoxigenin labeled probes using BCIP-NBT substrates . The tip1. - 2.tissue collection, fixation, and dehydratation as described in the procedure for the miRNA WISH. Proceed with 3 - 11.Permeabilization steps are performed as in the miRNA WISH procedure but omitting steps 12–14. The ! NOTE: Although the EDC cross-links the small RNA 5′ ends to the protein matrix, which has a superior sensitivity for miRNA-WISH, it is no longer needed in the RNA probes in the mRNA-WISH.12.hybridization as in the miRNA WISH with a hybridization temperature of 55°C and a probe concentration of 0.5 ng/μl. Perform the 13. Wash the probes in 50% formamide in 2×SSC + 0.1% Tween once for 10 min and once for 30 min at 55°C.14. Wash in 2×SSC twice for 5 min at RT.15. Wash in 0.2×SSC for 30 min at 55°C.! NOTE: During the incubation in 0.2×SSC, the probes should be shaken from time to time OR the incubation can be carried out on a shaker at 55°C.16. Wash twice for 10 min in the RNase buffer at RT.17. Incubate 15 min in RNase A (25 μg/ml in RNase buffer) at 37°C.18. Wash three times for 5 min in the RNase buffer at RT.! NOTE: The RNase buffer should be preheated to 37°C before use.19. Wash in 0.2×SSC for 30 min at 55°C.! NOTE: During the incubation in 0.2×SSC, the probes should be shaken from time to time OR the incubation can be carried out on a shaker at 55°C.20. Wash twice for 10 min in PBST.21. Incubate in 1% BSA in PBST for 90 min on a shaker at RT.22. Incubate with Anti-Digoxigenin-AP (1:1500) in 1% BSA in PBST for 4 h on a shaker at RT.⊕ PAUSE: The incubation with the antibody can proceed overnight at RT.23. Incubate in 1% BSA in PBST for 60 min on a shaker at RT.24. Wash three times for 10 min in PBST at RT.Probe detection, mounting, and microscope detection should be performed as was described in steps 25–30 for the miRNA-WISH procedure.Figure 1). Because of such heterogeneous cell populations, the results from RT-qPCR on whole explants are often difficult to interpret. Therefore, identifying the SE-associated genes requires insight into spatiotemporal expression patterns of the candidate genes. Hence, based on the original protocols for the direct localization of miRNA and mRNA using in situ hybridization with specific anti-sense LNA (locked nucleic acid) probes for miRNA molecules , while on the 10th day, the WISH signal was localized exclusively in the cotyledons that were engaged in the somatic embryo formation . Given that miR390 controls the lateral root development  and the proximity of SAM in SE-induced explants . The weak signal of miR167 corresponded to the results of the RT-qPCR analysis that showed a decreasing level of mature miR167 during SE culture . Similarly, a low level of miR167 during the induction stage of SE was observed in Longan .A signal for miR167 was barely detectable in the cotyledons , which did not correspond to the low amount of mature miR167 molecules that were found in the SE-induced explants using the RT-qPCR  and WISH  method.The reporter lines that have promoter fusions are the most common tools for the spatiotemporal analysis of the genes that are involved in SE induction , includiNA genes . Howeverelopment . AccordiMIM156 line, which has a decreased level of miR156 molecules . The obtained results showed that the miRNA WISH procedure can be used to detect the differences in miRNA abundance, which was previously shown in the roots and zygotic embryos of Arabidopsis (SPL (SQUAMOSA PROMOTER BINDING PROTEIN LIKE) genes have postulated to be among the candidate targets of miR156/157 in the SE of citrus , the signal of WISH miR166 was barely visible in the SE-induced explants of STTM165/166 , in comparison to strong signal of miR166 during SE culture in WT . In conclusion, the use of the clearing solution instead of 70% glycerol might be recommended in order to increase the detection of the low-abundant RNAs, in particular, when relatively thick samples of in vitro cultured tissue are being analyzed.Additionally, we evaluated the usefulness of the WISH method for detecting miRNAs with a low abundance in the explants. To this end, we performed WISH for the miR166 in the degraded . Consistre in WT . In ordere in WT . We founFigure 6). A strong miR390 signal was detected in the subprotodermal explant tissue from which the somatic embryos might be formed  as was also found by a cytological analysis .The development of somatic embryos is often overlooked and significantly more attention is paid to the induction phase of SE. Several studies have shown a similarity in the somatic and zygotic embryos in terms of their morphological, histological, biochemical, and physiological aspects, and have introduced SE as a model for studying ZE . Howeveranalysis . In lineanalysis  and soybanalysis , a WISH Figure 6D). Given that miR390 controls the lateral root development .Interestingly, somatic embryos that were at the torpedo stage of development displayed numerous isolated and intense miR390 signals that were located in the surface layer of the somatic embryo tissue  together with the accumulation of miR390 in the somatic embryos of different species, including longan, , citrus WOX5 (WUSCHEL-RELATED HOMEOBOX5), which is expressed in the quiescent center in embryos and mature roots . Consistent with the observation of WOX5 expression preceded the development of somatic embryos by the embryogenic tissue, we detected an intense WOX5 signal in the SE-involved cotyledons that were induced for 10 days . Then, the WISH for PHABULOSA (PHB), which is a target gene for the miR165/166 molecules  and sense  probes. The signal of the antisense PHB probe was detected in the cotyledons of 0 d explants  and in the proximity of SAM and vessels in the hypocotyl of 5 days explants . In a more advanced SE culture (10 days), an intense PHB signal was found in the SE-involved upper parts of the explant including in the proximity of SAM and cotyledons . However, we assumed that the hypocotyl-localized WISH signal might be a false positive background, as it was also detected by the PHB sense probe . The spatiotemporal pattern of the PHB transcripts corresponded to the one that is observed during ZE in Arabidopsis  are sensitive, efficient, and time saving as they do not require the long, time-consuming steps of preparing and sectioning tissues before hybridization. Thus, the recommended protocol enables the WISH signal to be monitored at different stages of a SE culture including the early and more advanced stages of SE in which the somatic embryos develop. The high sensitivity and specificity of the implemented miRNA-WISH with DIG-labeled LNA probes was proven by the very weak/absence of signal when using either the sense probes, animal miRNA (miR124) or when performing hybridization in the transgenic lines that had a reduced miRNA abundance.The protocols that are presented here, which enable the spatio-temporal localization of miRNAs and mRNAs in AW, MG, and MN conceived and designed the research. AW and MM optimized the protocols. AW conducted the experiments. AW, MM, MN, and JK contributed to the imaging using an Olympus BX-43 microscope, Pannoramic FLASH 250 II scanning microscope, reagents, and SEM, respectively. AW and MG analyzed the data and wrote the manuscript. All authors read and approved the manuscript.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.


Imatinib is an effective anticancer drug for the treatment of leukemia. Interestingly, when an FDA-approved drug library was tested for agents that block peroxisome proliferator-activated receptor γ (PPARγ) phosphorylation at Ser245 to evaluate possibilities of antidiabetic drug repositioning, imatinib was determined as a PPARγ antagonist ligand. However, it is not well understood how imatinib binds to PPARγ or would improve insulin sensitivity without classical agonism. Here, we report the crystal structure of the PPARγ R288A mutant in complex with imatinib. Imatinib bound to Arm2 and Arm3 regions in the ligand-binding domain (LBD) of PPARγ, of which the Arm3 region is closely related to the inhibition of PPARγ phosphorylation at Ser245. The binding of imatinib in LBD induced a stable conformation of helix H2′ and the Ω loop compared with the ligand-free state. In contrast, imatinib does not interact with Tyr473 on PPARγ helix H12, which is important for the classical agonism associated with side effects. Our study provides new structural insights into the PPARγ regulation by imatinib and may contribute to the development of new antidiabetic drugs targeting PPARγ while minimizing known side effects. Peroxisome proliferator-activated receptor γ (PPARγ) belongs to the thyroid hormone receptor-like nuclear receptor subfamily 1, which is one of the ligand-activated transcription factors . Through3 and is known to be activated by numerous endogenous and synthetic ligands ..Kd) of 1TZDs, such as rosiglitazone and pioglitazone, are potent insulin sensitizers for the treatment of type 2 diabetes mellitus . Howeverp-methoxyphenol group of lobeglitazone stabilized the hydrophobic pocket near the alternate binding site, which is formed by Ile249, Leu255, Ile281, Ile341, and Met348 . I. I35]. Ig assays . Interesg assays . These aIn conclusion, we have shown that imatinib binding strengthens the hydrophobic interaction network and stabilizes the surrounding helix H2′, the Ω loop, and the four-stranded β-sheet. This confirms the mechanism of ligand binding in the hydrophobic pocket of the Arm3 region affects the inhibition of Cdk5-mediated phosphorylation of PPARγ. In addition, our study provides a structural explanation as to why imatinib binding does not induce classical transcriptional activity of PPARγ. We expect our structural study to shed light on the development of a new generation of PPARγ ligands as antidiabetic drugs.Escherichia coli Rosetta 2(DE3) strain. A mutation of human PPARγ WT LBD at Arg288 (R288A) was generated using the QuikChange Site-Directed Mutagenesis Kit  and the mutation was confirmed by DNA sequencing.The cloning, expression, purification, and crystallization of human PPARγ LBD were mainly performed as previously reported . In bried-thiogalactopyranoside, the cells were further cultured for 20 h at 18 °C. The cells were harvested by centrifugation and lysed by sonication in buffer A  glycerol and 0.1 mM tris(2-carboxyethyl) phosphine hydrochloride) containing 5 mM imidazole and 1 mM phenylmethylsulfonyl fluoride. The suspension was centrifuged for 1 h, and the supernatant was filtered using a 0.22 μm syringe filter. The filtrate was applied onto a HiTrap Chelating HP column , which was pre-equilibrated with buffer A containing 5 mM imidazole. Upon applying a linear gradient of imidazole in the same buffer, PPARγ WT LBD was eluted at 50–100 mM imidazole concentration. The eluent was desalted in buffer A using a HiPrep 26/10 column  before cleaving the purified protein with 2 units of thrombin  per mg of PPARγ LBD. After cleavage with thrombin, both the N-terminal fusion tag and the uncleaved protein were removed on a HiTrap Chelating HP column . The flow-through was applied to a HiLoad XK-16 Superdex 200 prep-grade column , which was pre-equilibrated with buffer A. The purified PPARγ WT LBD was pooled and concentrated to 15.6 mg/mL using an Amicon Ultra-15 Centrifugal Filter Unit . The human PPARγ R288A mutant LBD was expressed and purified in the same manner as the WT protein.The recombinant PPARγ WT LBD cells were cultured in the Luria–Bertani medium containing 30 μg/mL kanamycin at 37 °C until the mid-log phase. After the addition of 0.5 mM isopropyl β-The purified PPARγ R288A mutant LBD and the coactivator SRC-1 peptide containing the LxxLL motif were mixed in a molar ratio of 1:2, in the presence or absence of a 7-fold molar excess of the PPARγ phosphorylation site inhibitor imatinib. After 24 h, the protein complexes were crystallized at 23 °C using the sitting-drop vapor diffusion method by mixing 0.2 μL of the purified protein solution and 0.2 μL of the reservoir solution. Crystals of imatinib-bound PPARγ R288A mutant LBD and ligand-free PPARγ R288A mutant LBD were obtained with a reservoir solution of 2.2 M sodium malonate at pH 7.0.HKL2000 [1212P2, with unit cell parameters of a = 130.9 Å, b = 52.8 Å, c = 53.4 Å. One monomer was present in the asymmetric unit, generating a Matthew’s parameter and solvent fraction of 2.70 Å3/Da and 54.5%, respectively. Crystals of ligand-free PPARγ R288A mutant LBD belonged to the space group 1212P2, with unit cell parameters of a = 131.5 Å, b = 52.7 Å, c = 53.7 Å. One monomer was present in the asymmetric unit, generating a Matthew’s parameter and solvent fraction of 2.72 Å3/Da and 54.9%, respectively. Data collection statistics are summarized in X-ray diffraction data for imatinib-bound PPARγ R288A mutant LBD were collected at 100 K using a Dectris PILATUS 2M-F pixel detector  at the NE3A experimental station of Photon Factory, Japan. The X-ray data from the crystal of ligand-free PPARγ R288A mutant LBD were collected at 100 K using a Quantum Q270 CCD detector system  at the BL-7A experimental station of Pohang Light Source, Korea. Raw X-ray diffraction data were processed and scaled using the program suit HKL2000 . CrystalMolRep with the ligand-free PPARγ WT LBD structure (PDB ID: 6JQ7) as a search model [COOT [REFMAC5, including the bulk solvent correction [freeR [MolProbity [Both structures of imatinib-bound and ligand-free PPARγ R288A mutant LBD were solved by the molecular replacement method using the program ch model ,37. Subsel [COOT . Followirrection . A totaln [Rfree . The rellProbity . RefinemKd) using Biacore T200 evaluation software 3.0 .The binding affinities of PPARγ WT LBD and R288A mutant LBD with imatinib were investigated by SPR kinetics experiments. All SPR measurements were performed at 25 °C using the Biacore T200 apparatus . For immobilization, the amine coupling kit containing 0.1 M N-hydroxysuccinimide and 0.4 M 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride on a CM5 sensor chip with HBS-EP buffer  was used according to the manufacturer’s protocol. Subsequently, 30 μg/mL of PPARγ WT LBD or R288A mutant LBD dissolved in 10 mM sodium acetate at pH 5.0 was injected at regular intervals until the immobilization level reached approximately 7000 response units (RU). The remaining activated carboxyl groups on the CM5 sensor chip surface were deactivated with 1 M ethanolamine at pH 8.5 for 400 s. The control experiment was treated identically with a reference flow cell without protein, and the response by the control was subtracted from each sample dataset. Imatinib at concentrations of 0.625, 1.25, 2.50, 5.00, and 10.0 μM in PBS buffer was injected over the PPARγ WT LBD chip or R288A mutant LBD chip at a rate of 30 μL/min for 120 s, followed by dissociation for 300 s in multi-cycle reactions. For rosiglitazone, experiments were conducted at lower concentrations than imatinib. The sensor chip surface was regenerated for 5 s with 5 mM NaOH between cycles. The SPR response data were fit to the simple bimolecular 1:1 Langmuir isotherm binding model to determine the equilibrium dissociation constant (Atomic coordinates and structure factors for ligand-free and imatinib-bound PPARγ R288A mutant LBD structures have been deposited in Protein Data Bank under the accession codes 6KTM and 6KTN, respectively.


Physcomitrella. We find that this newly identified role for CLAVATA in regulating cell division plane orientation is shared between Physcomitrella and Arabidopsis. We report that roles for CLAVATA in regulating cell proliferation and cell fate are also shared and that CLAVATA-like peptides act via conserved receptor components in Physcomitrella. Our results suggest that CLAVATA was a genetic novelty enabling the morphological innovation of 3D growth in land plants.How genes shape diverse plant and animal body forms is a key question in biology. Unlike animal cells, plant cells are confined by rigid cell walls, and cell division plane orientation and growth rather than cell movement determine overall body form. The emergence of plants on land coincided with a new capacity to rotate stem cell divisions through multiple planes, and this enabled three-dimensional (3D) forms to arise from ancestral forms constrained to 2D growth. The genes involved in this evolutionary innovation are largely unknown. The evolution of 3D growth is recapitulated during the development of modern mosses when leafy shoots arise from a filamentous (2D) precursor tissue. Here, we show that a conserved, CLAVATA peptide and receptor-like kinase pathway originated with land plants and orients stem cell division planes during the transition from 2D to 3D growth in a moss,  •CLAVATA originated in the last common ancestor of land plants•PhyscomitrellaCLAVATA regulates cell proliferation, fate, and growth in •Physcomitrella and ArabidopsisCLAVATA orients cell division planes in •Physcomitrella and ArabidopsisCLEs act via receptors that are conserved between  Whitewoods, Cammarata, et al. show that a conserved CLAVATA (CLV) pathway arose in the last common ancestor of land plants. CLV regulates cell division plane orientation during the 2D to 3D growth transition in a moss, and roles for CLV are shared between mosses and flowering plants, suggesting that CLV enabled 3D growth to arise in land plants. The conquest of land was enabled by a series of innovations that allowed plant forms to radiate and occupy new volumes of space in the sub-aerial environment . Among tArabidopsis, the CLAVATA (CLV) and WUSCHEL (WUS) pathways act in a feedback loop to regulate many aspects of stem cell function, including cell fate [CLV3 encodes a small, secreted peptide that is expressed in the upper cell layers of the central zone and can move throughout the meristem [CLV1 is expressed in the underlying cell layers of the central zone and encodes a receptor-like kinase that acts as a receptor for CLV3 [WUS activity promotes meristem cell proliferation [WUS expression domain [CLV3 expression [In ell fate , 9, prolell fate , 11, andell fate . CLV3 enmeristem , 14, 15.for CLV3  in conjufor CLV3 , 18. WUSferation , and CLVn domain . WUS actpression , therebyPhyscomitrella stem cell function, we first queried publicly accessible genome and transcriptome databases from a wide range of green algae and land plants for CLV3-like (CLE), CLV1/BAM, RPK2, CLV2, and CRN homologs -like CLEs that regulate vascular development in Arabidopsis, Physcomitrella does not, consistent with an evolutionary loss in mosses . One RPK2 homolog was found and named PpRPK2, but no CLV2 or CRN homologs were found. These sequence data indicate that the core components of the CLV pathway first arose in the last common ancestor of land plants, alongside the evolutionary innovation of 3D growth [To determine how the CLV pathway evolved and identify potential roles for CLV in homologs . We founivergent . The genn mosses ; Data S1n mosses  and S3. volution , therebyD growth .Physcomitrella CLV activity, we first analyzed gene expression patterns in relation to the transition between 2D filamentous and 3D gametophore growth  fusion lines for PpCLE1, PpCLE2, PpCLE7, PpCLV1a, PpCLV1b, and PpRPK2 as RT-PCR showed that these 6 genes were upregulated at around the time of gametophore initiation  plants  to silence expression of th phase A–3E. How) plants U, and Ppelopment U. These elopment F–3T. Alt compare G and 3H. compare L and 3M. compare Q and 3R. mutants S. Pprpk2 defects T. The muts e.g., L and 3M Arabidopsis. To this end, we examined Arabidopsis clv1/bam1/bam2/bam3 quadruple mutant meristems, in which the function of the entire CLV/BAM gene clade is lost [clv1/bam1/bam2/bam3 mutant roots  mutants. However, Physcomitrella does not fit the Arabidopsis paradigm of meristem function because the shoot apex comprises a single apical stem cell. The apical cell cleaves merophyte daughter cells in a spiral pattern, and merophytes subsequently divide to generate leaf initials and stem tissues [Physcomitrella and Arabidopsis, we imaged one of the largest gametophores from 1-month-old WT and mutant plants using light and confocal microscopy and found that mutant gametophores were reduced in height and had developmental defects  have no appreciable effect on gametophore development, Arabidopsis CLV3 and all of the Physcomitrella CLEs cause gametophore dwarfing and a strong reduction in leaf size correlating with a reduction in leaf cell number  medium plates containing solute or peptides at a 1 μM concentration. Although solute controls, a randomized peptide, and CLE41 caused no arrest of root development, CLV3 and all of the Physcomitrella CLEs caused a significant reduction in root length in Arabidopsis resulting from collapse of the root meristem , are known to specify cell division planes in  embryos , and auxand NOG1 , 31. Themitrella , 33, 34,erturbed  and 5 anerturbed , and thePpcle mutants specifically affect the transition to 3D growth and the 3D growth phase, and morphological responses to peptide application are also specific to the 3D growth phase. Thus, in an ancient land plant group, CLV regulates a developmental transition that mirrors an evolutionary transition. The data suggest that CLV was a genetic novelty for a key morphological innovation of land plants.The data we present are important in two evolutionary contexts. First, they show that the CLV pathway originated with land plants and that CLV-regulated stem cell proliferation and function is likely to be an ancestral feature of land plants. The acquired capacity of land plants to orient stem cell divisions in multiple planes enabled diversification by permitting plants to develop upright axes with organs arranged in multiple orientations, a crucial step in shoot evolution . Stem ceKNOX genes regulate meristematic proliferation in vascular plants [KNOX (MKN) genes are primarily expressed in sporophyte tissues [mkn2 mutants have elongation defects in sporophytes, they have normal gametophytes [WOX genes are key regulators of stem cell proliferation in Arabidopsis [WUS gene clade [Physcomitrella are likely to be distinct from those in Arabidopsis as Ppwox13L mutant gametophores develop normally [KNOX- and WOX-regulated meristem functions were both acquired after the bryophyte-vascular plant divergence. CLV was important in the origin of land plant meristem functions in the gametophyte stage of the life cycle, and we speculate that CLV was recruited to regulate stem cell function in the sporophyte stage of the life cycle prior to the origin of KNOX- and WOX- regulated meristem functions.Second, the data are important in the context of evolving gene regulatory networks for land plant meristem function. Whereas the first land plant meristems comprised a single gametophytic stem cell, the multicellular sporophyte meristems of vascular plants combine stem cell and more generally proliferative capacities . Class Ir plants , 36, but tissues , 37, andbidopsis . Howeverne clade , 39, andnormally . Thus, cjill.harrison@bristol.ac.uk). Please note that the transfer of transgenic materials will be subject to MTA and any relevant import permits.Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Jill Harrison (rpk2-4 (cli) or clv1/bam1/bam2/bam3 mutants [Arabidopsis experiments. Homozygous rpk2-4 mutants were confirmed using a BamHI dCAPs screen with a PCR fragment amplified using primers AtRPK2-BamHIF and AtRPK2-BamHIR (see primer list). Seeds were surface sterilized in 5% (v/v) sodium hypochlorite for 10 min and washed three times with sterile de-ionised water. They were then stratified at 4°C in darkness for 48 hr and sown on 0.5 X MS plates containing 0.8% agar [rpk2 experiments) or at 22°C under continuous light (clv1/bam1/bam2/bam3 experiments).Columbia (Col-0),  mutants , 25 were.8% agar . Plants Physcomitrella patens [-1 in Sanyo MLR-351 growth cabinets. BCDAT medium comprises 250mg/L MgSO4.7H2O, 250mg/L KH2PO4 (pH6.5), 1010mg/L KNO3, 12.5mg/L, FeSO4.7H2O, 0.001% Trace Element Solution 2.12H2O, 0.055mg/L CuSO4.5H2O, 0.028mg/L KBr, 0.028mg/L LiCl, 0.389mg/L MnCl2.4H2O, 0.055mg/L CoCl2.6H2O, 0.055mg/L ZnSO4.7H2O, 0.028mg/L KI and 0.028mg/L SnCl2.2H2O), 0.92 g/L C4H12N2O6 and 8g/L agar with CaCl2 added to a 1mM concentration after autoclaving. Protonemal cultures for transformation were grown on BCDAT plates overlaid with autoclaved cellophane disks and molecular and phenotypic analyses were undertaken using 1 mm spot cultures unless otherwise stated.The Gransden strain of a patens  was usedArabidopsis thaliana and Oryza sativa CLE sequences were respectively retrieved from TAIR and RAP-DB [Selaginella moelendorffii [Glycine max [Picea abies [CLEs were retrieved from NCBI. To extend taxon sampling within land plants and identify previously unknown CLEs, the CLE domains of Arabidopsis thaliana CLV3 and CLE41 were used as tBLASTn queries with an e-value cutoff of e-100 to screen transcriptome or draft genome assemblies of a basal angiosperm (Amborella trichopoda), a fern (Diplazium wichurae), a hornwort (Anthoceros agrestis), a moss (Physcomitrella patens v1.6 [Marchantia polymorpha). Positive hits were used in reciprocal BLASTs until no new sequences were retrieved. All sequences retrieved were checked for the presence of a signal peptide [Physcomitrella genome  and chlorophyte algae  were also searched but no CLEs were found. A previously annotated Chlamydomonas reinhardtii CLE [CLEs and a premature in-frame stop codon. A full list of taxa and databases searched is given in Previously described d RAP-DB . Selaginndorffii , Glycinecine max , 58 and ea abies  CLEs werens v1.6 ) and a l peptide  using Si peptide , 62. New peptide  succeede peptide . An updae (v 3.1 ) also sudtii CLE  was re-aArabidopsis CLV1 and RPK2 sequences were used to query the databases listed above using tBLASTn searches with an e-value cut-off of e-1000. As the LRR-Receptor kinase family is large, only sequences that retrieved CLV1 or RPK2 as a top hit in reciprocal BLASTs to Arabidopsis were used in further analyses. Newly identified CLV1-like and RPK2-like genes were named with a two-letter prefix denoting the genus and species and given an alphabetical epithet . To infGA7.0.26   as a loading control. Where possible, primers were designed to span introns to detect genomic contamination, and sequences are listed in Total RNA was isolated from 4 day-old protonemal cultures and 10, 21 or 28 day old spot cultures using the QIAGEN RNeasy method. RNA was DNase treated prior to reverse transcription with SuperScript II following manufacturer’s guidelines. Semiquantitative RT-PCR was undertaken using 2EDTA.Genomic DNA was extracted from protonemal cultures using a CTAB (Hexadecyltrimethylammonium bromide) protocol. Snap-frozen tissue was ground in liquid nitrogen and transferred to tubes containing prewarmed extraction buffer , with no more than 100 mg of tissue per mL of buffer. Samples were incubated for 10 min at 65°C and an equal volume of 24:1 chloroform:isoamyl alcohol was added and mixed with each sample to form an emulsion. The tubes were centrifuged at high speed  for 10 min, and the aqueous phase was transferred to a fresh tube prior to DNA precipitation with an equal volume of isopropanol and repeated centrifugation. DNA was washed with 70% ethanol and dissolved in water, 10 mM Tris pH 8.0 or 10 mM Tris pH 8.0 with 1 mM NaPpCLE1 (2.1 kbp), PpCLE2 (2.1 kbp), PpCLE7 (2 kbp), PpCLV1a (2 kbp), PpCLV1b (2.8 kbp) and PpRPK2 (1.4 kbp) were PCR amplified using a proof-reading Taq polymerase and primers listed in Promoter sequences from PplcleAmiR1-3 and PplcleAmiR4-7 constructs, resistance cassettes from pGREEN [UBIQUITIN promoter from pBRACT211 [To generate m pGREEN  were firm pGREEN . A soybeBRACT211  was inseBRACT211 , generatPhyscomitrella U3 or U6 promoter and flanked by attB sites were synthesized and cloned into pDONR201. sgRNA sequences were selected and screened for off target hits in the Physcomitrella V3 genome using http://crispor.tefor.net/. To clone guide RNAs into expression cassettes, two primers consisting of guide sequences with overhangs for U3 and U6 promoters were annealed and ligated into U3 or U6 expression vectors pre-digested with BsaI. Constructs were checked by sequencing and co-transformed with pACT::Cas9 [Small cassettes containing two BsaI restriction sites and sgRNAs  driven bCT::Cas9  to enginPpRPK2 flanking regions. This plasmid was checked by sequencing and linearized for transformation as illustrated in 5′ and 3′ flanking regions were PCR amplified with a proof-reading Taq polymerase and cloned sequentially into pGEMT-EASY using primers listed in 3 solution , 10 mM Tris pH7.2) to 2 g of molten PEG 6000, and the tube containing the solution was left in a water bath at 45°C. To isolate protoplasts, homogoenous protonemal cultures were grown for 5 days to a week post passage. A 1% driselase solution was prepared in 25 mL 8% mannitol, and the supernatant was removed and filter sterilized into to a clean 50 mL falcon tube following centrifugation. Tissue from 4-6 plates was transferred into the driselase solution and the tissue suspension was left for 30-40 min with intermittent mixing to allow cell wall digestion. The mixture was then transferred into a fresh tube through a 50 μm filter to remove cell and cell wall debris. Protoplasts were sedimented by centrifugation for 3 min at 120 g, resuspended and washed three times in 10 mL of 8.0% mannitol prior to counting with a hematocytometer. Protoplasts were then sedimented and resuspended to a density of 1.2 × 106 per mL in MMM solution . 300 μL aliquots of protoplasts were dispensed into falcon tubes prior to addition of DNA and 300 μL PEG solution, and cells were then heat shocked for 5 min at 45°C. Transformation mixtures were progressively diluted with 1 mL of 8% mannitol solution and washed. Protoplasts were then sedimented by centrifugation as above and washed four more times. After the final wash and spin, protoplasts were resuspended in 5 mL liquid BCD medium (constituents as specified above but without ammonium tartrate or agar) with 8% mannitol, 10 mM CaCl2 and 0.5% glucose, wrapped in aluminum foil and left at 23°C overnight. The next day, the protoplast suspension was plated onto BCDAT plates overlain with cellophane and containing 8% mannitol and 5 g/L glucose, using c.1 mL per plate. Plants were grown under standard conditions until regenerants comprised 10-20 cells. Cellophane discs were then transferred onto BCDAT plates containing antibiotics for selection . Plants were grown for 2 weeks on selection plates prior to transfer onto BCDAT plates lacking antibiotic for 2 weeks and then back on to selection plates for a further 2 weeks. All lines were screened by PCR, RT-PCR, Southern analysis or sequencing as illustrated in For gene targeting and AmiR approaches, 10-20 μg of plasmid DNA was isolated using the QIAGEN Plasmid Plus Midi system and linearized as illustrated in PpcleAmiR Southerns, 10-15 μg genomic DNA was digested with EcoRV and fractionated in 0.8% agarose by gel electrophoresis. DNA in each gel was depurinated with 0.2 M HCl for 20 min and denatured with 0.4 M NaOH for 20 min prior to neutralization for 20 min in a solution containing 3 M NaCl and 1 M Tris pH 7.5. Gels were inverted onto a Whatman paper wick inserted into a bath of 20 X SSC solution, and DNA was transferred onto a nitrocellulose membrane by overnight Southern blotting. DNA was UV crosslinked to the membrane and the membrane was rinsed in water prior to immersion in pre-hybridization solution . The probe template was excised with EcoRV and BamHI from the PpcleAmiR1-3 construct and the probe was synthesized using an Amersham Rediprime II DNA labeling kit as per manufacturer’s instructions. Hybridization was undertaken in a 3 X SSC buffer at 58°C and this was followed by two 20 min washes at 58°C in 3 X SSC and 2 X SSC buffers respectively. Membranes were wrapped in Saran Wrap and used to expose X-ray film, and film was then developed using a film processor. For promoter::NGG and Pprpk2 Southerns, 2.5-3 μg genomic DNA was digested as illustrated in PIG1 flanking sequence, PpRPK2 coding sequence or a hygromycin resistance cassette were PCR amplified and labeled using the Roche DIG High Prime system. Hybridization was undertaken overnight at 42°C using the Roche DIG Easy Hyb system. Washing and detection were performed using the manufacturer’s protocol from the Roche DIG High Prime DNA labeling and Detection Starter kit II.For To assess whole plant and gametophore phenotypes, 4 to 5 week-old spot cultures were imaged using a Keyence VHX-1000E digital microscope with a 20-50 X or 50-200 X objective. To analyze bud phenotypes, confocal imaging was undertaken on tissue stained with 0.5 mg/ml propidium iodide using a Leica TCS SP5 microscope with excitation from the 488 or 514 laser line and emission collected at 600-650 nm or using a Zeiss 710 LSM with excitation from a 514 laser line and emission collected at 566-650 nm. To analyze leaf phenotypes, leaves were removed form gametophores, arranged in heteroblastic series, cleared in 1% chloral hydrate overnight, washed in deionised water and treated with 2M NaOH for 2 hr. They were then washed with water and stained with 0.05% toluidine blue for 2 min before destaining for 10 min in water. The stained leaves were then mounted on a slide under a coverslip and imaged to visualize cell outlines. Adobe Illustrator was used to trace leaf outlines to produce silhouettes for illustration purposes . Quantitrpk2 meristems, roots were stained with Lugol’s stain, cleared, and imaged using a 20 X objective on a Leica DMRXA microscope with DIC [clv1/bam1/bam2/bam3 roots were stained with 15 mM propidium iodide and imaged using a C-Apochromat 40 X/1.20 W Korr objective on a Zeiss LSM710 microscope. Excitation and emission windows for propidium iodide were 560 nm and 566-719 nm respectively. Confocal images were analyzed and processed using ImageJ and Adobe Photoshop.Root length was scored from scanned images of plants grown on ½ X MS plates using ImageJ . To visuwith DIC . clv1/baPhyscomitrella plants grown on BCDAT were cut out of plates with agar and incubated at 37°C in a 100 mM phosphate buffer with 10 mM Tris pH8.0, 1 mM EDTA pH8.0, 0.05% Triton X-100, 1 mg/mL X-GlcA (5-Bromo-4-chloro-3-indolyl-β-D-glucuronic acid) and potassium ferri/ferrocyanide using concentrations and times indicated in Synthetic CLE peptides  were dissolved in phosphate buffer  to stock concentrations of 1 mM and 10 mM. Plants were grown on BCDAT plates containing peptides diluted to concentrations specified in the main text.Quantification and statistical analyses were undertaken as stipulated in main text and SI figures and figure legends.Genome and transcriptome data were searched as described in


Members of the transient receptor potential (TRP) ion channels conduct cations into cells. They mediate functions ranging from neuronally mediated hot and cold sensation to intracellular organellar and primary ciliary signaling. Here we report a cryo-electron microscopy (cryo-EM) structure of TRPC4 in its unliganded (apo) state to an overall resolution of 3.3 Å. The structure reveals a unique architecture with a long pore loop stabilized by a disulfide bond. Beyond the shared tetrameric six-transmembrane fold, the TRPC4 structure deviates from other TRP channels with a unique cytosolic domain. This unique cytosolic N-terminal domain forms extensive aromatic contacts with the TRP and the C-terminal domains. The comparison of our structure with other known TRP structures provides molecular insights into TRPC4 ion selectivity and extends our knowledge of the diversity and evolution of the TRP channels. Members of the transient receptor potential (TRP) ion channels conduct cations into cells upon activation by a variety of signals. Here authors present the cryo-EM structure of TRPC4 in its unliganded (apo) state, which provides molecular insights into TRPC4's ion selectivity and TPR channel evolution. The TRPC subfamily are non-selective cation channels  that alter proliferation, vascular tone, and synaptic plasticity3. This family can be further subdivided into two subgroups: TRPC2/3/6/7 and TRPC1/4/5. TRPC4 is broadly expressed in human tissues and can assemble as homomeric channels or form heteromeric channels with TRPC1 and TRPC57. Studies of Trpc4-deficient mice have shown that TRPC4 affects endothelial-dependent regulation of vascular tone, endothelial permeability, and neurotransmitter release from thalamic interneurons8. Stimulation of Gq and Gi/oG-protein-coupled receptors (GPCRs) as well as tyrosine kinase receptors potentiate channel activity10. Activation of TRPC4 is regulated by intracellular Ca2+, phospholipase C, and membrane lipids by unclear mechanisms. In addition, Storch et al.11 have proposed a potential mechanism related to phosphatidylinositol 4,5-bisphosphate and Na+/H+ exchanger regulatory factor proteins11.Mammalian transient receptor potential (TRP) channels are activated by a wide spectrum of ligands, temperature, lipids, pH, and as yet unknown stimuli. They are classified into six subfamilies based on sequence similarity: TRPC , TRPM (melastatin), TRPV (vanilloid), TRPA (ankyrin), TRPML (mucolipin), and TRPP (or PKD) (polycystin)17, TRPA118, TRPP119, TRPML120, TRPM424, and TRPM825 have been solved. Here we present the structure of mouse TRPC4 in its apo state at pH 7.5 at an overall resolution of 3.3 Å. The structure provides detailed information on the ion permeation, selectivity, and gating mechanism of TRPC subfamily.Along with the revolution in cryo-electron microscopy (cryo-EM), improved sample preparation, data acquisition, and image processing strategies, the structures of TRPVsThe mouse TRPC4 (residues amino acids (a.a.) 1–758, excluding a.a. 759–974) was expressed using the BacMam expression system (Methods) and purified protein (pH 7.5) was used for single-particle cryo-EM analysis  was permeable to cations and retained sensitivity to channel activator englerin A, blocker ML204, and blocker 2-APB  relationships from both constructs were typical of TRPC4/5, with flattening of the curve between 10 and 40 mV due to outward Mg2+ block26. These results suggest that, in the context of chemical modulation, the biophysical properties of the truncated construct are similar to that of the full-length TRPC4.Calls Fig. . In patclls Fig. . The curq proteins), extracellular application of ADP (P2Y1 agonist) or ATP (P2Y2 agonist) induced large currents for full-length TRPC4. In contrast, the currents for truncated TRPC4 were much smaller but still exhibited the characteristic I–V relationship of the TRPC4 channel  or carbachol (M2R)  connects the pre-S1 helix with the pre-S1 elbow  could be activated by englerin A but not DTT  and cysteine mutants of TRPC4. Reducing agents dithiothreitol  and tris(2-carboxyethyl)phosphine hydrochloride  potentiated wt TRPC4 whole-cell currents Fig. . The I–VDTT Fig. , suggestDTT Fig. . If thisn  and cation–π interactions  is Val961 at its lower gate18  is conserved in all the TRPC channels  and some divalents, such as Ca2+. A strong non-protein density peak in our TRPC4 structure is present in a hydrophilic pocket on the cytoplasmic side of the S1–S4 fold, consistent with the corresponding location of a presumed Ca2+ in TRPM421  and phospholipids (the density fitting ceramide-1-phosphate or phosphatidic acid) .TRPC4 has been characterized as a non-selective cation channel with moderate selectivity for Ca4. This phenomenon can be explained in our structure, by the pore helix located at the LFW motif , which is conserved in all TRPC members, results in a dominant-negative channel34. However, the results of TRPC5 cysteine mutations are disparate. Xu et al.33 reported that mutation of either a single cysteine  or both cysteines (C553A+C558A) resulted in constitutively active TRPC5 channels. Moreover, the disulfide bond was formed between C553 and C558 in the same monomer and not crosslinked between different monomers. These observations were challenged by a later study stating that both C553S and C558S mutants completely lost channel activity, and dimerization of channel proteins was significantly impaired for cysteine mutants of both TRPC4 and TRPC534.The role of the disulfide bond in TRPC4’s pore loop has not been previously investigated, but electrophysiological studies on the closely related TRPC5 have shown that reduction of the disulfide bond with DTT potentiated TRPC5 currentsOur structural results demonstrate the existence of a disulfide bond in the pore region. If one of the two cysteines is mutated, an inter-monomer disulfide bond may be formed which would substantially change the architecture of the pore loop and affect channel activity. This is supported by our electrophysiological observations from the C549A and C554A TRPC4 mutants. Specifically, results of the double cysteine mutant (C549A+C554A) suggest that the pore loop disulfide bond is dispensible for channel activation by the activator, but is essential for redox regulation of channel activity.q protein-coupled receptors and increasing intracellular Ca2+ as well as by PIP2 hydrolysis, while TRPC3, 6, and 7 are additionally sensitive to diacylglycerol35. From our observations, we propose that our TRPC4 structure represents a closed state because of the narrow distance between the residues in the lower gate. In the present study, we truncated a.a. 759–974 from TRPC4, resulting in the absence of the PDZ-binding motif, a Gα-binding sites, the second camodulin-binding domain and a potential PIP2-binding region. We found that this truncation greatly impaired or abolished channel activity in response to Gq-mediated and Gi/o-mediated signaling, which confirmed the importance of the C terminus in receptor-operated activation of TRPC4. However, the truncated TRPC4 currents stimulated by P2Y1 and P2Y2 were small and thus there are other structural constraints related to Gq-mediated TRPC4 activation. The truncated TRPC4 retained the first camodulin-binding domain and thus could still be sensitive to intracellular Ca2+ changes, such Ca2+ release from the ER stores induced by IP3. Our current study should help guide future mutagenesis, functional, and structural studies.TRPCs have multiple activation mechanisms. TRPC1/4, 1/5 heterotetramers and TRPC4 or 5 homotetramers can be activated by G36 and a maltose-binding protein (MBP) tag was added to its N terminus  were infected with 10% (v/v) P3 baculovirus at a density of 2.0–3.0 × 106 cells/ml for protein expression at 37 °C. After 12–24 h, 10 mM sodium butyrate was added and the temperature reduced to 30 °C. Cells were harvested at 72 h after transduction, and resuspended in a buffer containing 30 mM HEPES, 150 mM NaCl, 1 mM DTT, pH 7.5, with EDTA-free protease inhibitor cocktail (Roche). After 30 min, cells were solubilized for 2–3 h in a buffer containing 1.0% (w/v) N-dodecyl-β-d-maltopyranoside (Affymetrix), 0.1% (w/v) CHS (Sigma), 30 mM HEPES, 150 mM NaCl, 1 mM DTT, pH 7.5, with EDTA-free protease inhibitor cocktail (Roche). The supernatant was isolated by 100,000 × g centrifugation for 60 min, followed by incubation in amylose resin (New England BioLabs) at 4 °C overnight. The resin was washed with 20 column volumes of “wash buffer” containing 30 mM HEPES, 150 mM NaCl, 0.1% (w/v) digitonin, 0.01% (w/v) CHS, 1 mM DTT, pH 7.5, with EDTA-free protease inhibitor cocktail (Roche). The protein was eluted with four column volumes of wash buffer with 40 mM maltose. The protein was then concentrated to 0.5 ml with a 100 kDa molecular weight cut-off concentrator (Millipore). PreScission protease was added to the samples and incubated overnight at 4 °C to remove the MBP tag. After incubation at 4 °C overnight, the protein was then purified on a Superose 6 column in a buffer composed of 30 mM HEPES, 150 mM NaCl, 0.1% (w/v) digitonin, 1 mM DTT, pH 7.5. The peak corresponding to tetrameric TRPC4 was collected and concentrated to 4.5 mg/ml for cryo-EM studies.The mouse TRPC4 construct (a.a. 1–758 of 974) was cloned into the pEG BacMam vector37, with a defocus range between 1.5 and 3.0 μm. The electron dose was set to 8 e−/physical pixel/s and the sub-frame time to 200 ms. A total exposure time of 10 s resulted in 50 sub-frames per image stack. The total electron dose was 52.8 e−/Å2 (~1.1 e−/Å2 per sub-frame).Purified TRPC4 protein (3.5 µl) in digitonin at 4.5 mg/ml was applied onto a glow-discharged, 400 mesh copper Quantifoil R1.2/1.3 holey carbon grid (Quantifoil). Grids were blotted for 7 s at 100% humidity and flash frozen by liquid nitrogen-cooled liquid ethane using a FEI Vitrobot Mark I (FEI). The grid was then loaded onto an FEI TF30 Polara electron microscope operated at 300 kV accelerating voltage. Image stacks were recorded on a Gatan K2 Summit (Gatan) direct detector set in super-resolution counting mode using SerialEM38. The images from the sum of all frames with dose weighting were subjected to visual inspection and poor images were removed before particle picking. Particle picking and subsequent bad particle elimination through 2D classification was performed using Python scripts/programs39 with minor modifications in the 8× binned images. The selected 2D class averages were used to build an initial model using the common lines approach implemented in SPIDER40 through Maofu Liao’s Python scripts39, which was applied to later 3D classification using RELION41. Contrast transfer function parameters were estimated using CTFFIND442 using the sum of all frames without dose weighting. Quality particle images were then boxed out from the dose-weighted sum of all 50 frames and subjected to RELION 3D classification. RELION 3D refinements were then performed on selected classes for the final map43. The resolution of this map was further improved by using the sum of sub-frames 1–14.Image stacks were gain normalized and binned by 2× to a pixel size of 1.23 Å prior to drift and local movement correction using motionCor2 44. Taking advantage of the defined geometry of helices and clear bumps for Cα atoms in the TMD, amino acid assignment was subsequently achieved based primarily on the clearly defined side chain densities of bulky residues. The refined atomic model was further visualized in COOT. A few residues with side chains moving out of the density during the refinement were fixed manually, followed by further refinement. The TRPC4 model was then subjected to global refinement and minimization in real space using the PHENIX45 module “phenix.real_space_refine”46 and geometry of the model was assessed using MolProbity47 in the comprehensive model validation section of PHENIX. The final model exhibited good geometry as indicated by the Ramachandran plot . The pore radius was calculated using HOLE48.For the TRPC4 structure, a polyalanine model was first built in COOT2, 0.03 CaCl2, 0.05 EGTA, 10 HEPES, and the pH was titrated to 7.2 using CsOH. The standard bath solution contained (mM): 140 NaCl, 5 KCl, 1 MgCl2, 2 CaCl2, 10 HEPES, 10 d-glucose, and the pH was adjusted to 7.4 with NaOH. The recording chamber had a volume of 150 µl and was perfused at a rate of ~2 ml/min. For Ca2+ imaging experiments, transfected 293T cells were seeded on coverslips and incubated with Fura-2 AM (2 μM) for 30 min at 37 °C in standard bath solution. The ratio (F340/F380) of Ca2+ dye fluorescence was measured by a Nikon Ti-E system with the NIS-Elements software. All the experiments were performed at room temperature.Mouse TRPC4α, TRPC4β, and μOR were cloned from mouse brain complementary DNA (cDNA) (primer sequences used in this study are in Supplementary Table https://www.ebi.ac.uk/pdbe/emdb/, and the fitted coordinate has been deposited in the Protein Data Bank, [PDB:5Z96] www.pdb.org.Data supporting the findings of this manuscript are available from the corresponding authors upon reasonable request. Data deposition: Cryo-EM electron density map of the mouse TRPC4 has been deposited in the Electron Microscopy Data Bank, [EMD:6901] Supplementary InformationPeer Review File


While the G6PD is the key enzyme of removing oxidative stress in erythrocytes, the early diagnosis is utmost vital to prevent chronic and drug-, food- or infection-induced hemolytic anemia. The characterization of the mutations is also important for the subsequent genetic counseling, especially for female carrier with ambiguous enzyme activities and males with mild mutations. While multiplex SNaPshot assay and Sanger sequencing were performed on 500 G6PD deficient males, five newly discovered variations, namely c.187G > A (p.E63K), c.585G > C (p.Q195H), c.586A > T (p.I196F), c.743G > A (p.G248D), and c.1330G > A (p.V444I) were detected in the other six patients. These variants were previously named as the Pingtung, Tainan, Changhua, Chiayi, and Tainan-2 variants, respectively. The  Putative mutations were confirmed by sequencing of an independent PCR product. The study protocol was reviewed and approved by the Institutional Review Board of Taipei City Hospital, Taiwan.In 500 G6PD-deficient male newborns detected by G6PD enzyme activity assay 2.2Conservation of the peptide sequence around the affected residues was assessed by alignment of orthologous and human G6PD sequences with ClustalW2, 2.3in silico analyses were performed using the SIFT Different online algorithms were used to predict the functional consequences of the five variants. The 2.4NM_001042351. The amino acid numbers were counted from the N-terminal Met of human G6PD protein.Distribution of alterations was highlighted in the coding region and the functional domains 2.5G6PD variations observed in this study were presented based on the X-ray crystal structure available at the Protein Data Bank from human G6PD protein (PDB code 1QKI) The 3D structure of 2.6When protein structure is important to predict the effects of variants


Under certain conditions, amyloid-like fibrils can develop into three-dimensional networks and form hydrogels by a self-assembly process. When Cu/Zn superoxide dismutase (SOD1), an anti-oxidative enzyme, undergoes misfolding, fibrillar aggregates are formed, which are a hallmark of a certain form of familial amyotrophic lateral sclerosis (ALS). However, the issue of whether SOD1 fibrils can be assembled into hydrogels remains to be tested. Here, we show that the SOD1 polypeptides undergo hydrogelation accompanied by the formation of thioflavin T-positive fibrils at pH 3.0 and 4.0, but not at pH 5.0 where precipitates are formed. The results of viscoelastic analyses indicate that the properties of SOD1 hydrogels (2%) were similar to and slightly more fragile than a 0.25% agarose gel. In addition, monitoring by a quartz crystal microbalance with admittance analysis showed that the denaturing of immobilized SOD1 on a sensor under the hydrogelation conditions at pH 3.0 and 4.0 resulted in an increase in the effective acoustic thickness from ~3.3 nm (a folded rigid form) to ~50 and ~100 nm (an extended water-rich state), respectively. In contrast, when SOD1 was denatured under the same conditions at pH 5.0, a compact water-poor state with an effective acoustic thickness of ~10 nm was formed. The addition of physiological concentrations of NaCl to the pH 4.0 sample induced a further extension of the SOD1 with larger amounts of water molecules (with an effective acoustic thickness of ~200 nm) but suppressed hydrogel formation. These results suggest that different denatured intermediate states of the protein before self-assembly play a major role in determining the characteristics of the resulting aggregates and that a conformational change to a suitable level of extended water-rich intermediate state before and/or during intermolecular assembling is required for fibrillation and hydrogelation in the case of globular proteins. Under certain conditions, fibrous proteins such as gelatin  and fibr2CG-NH2  can deve2CG-NH2 , islet a2CG-NH2 , were al2CG-NH2 , β-lacto2CG-NH2 , 8, bovi2CG-NH2  and α-sy2CG-NH2  also are2CG-NH2 , 12. Hyd2CG-NH2 . TherefoSOD1 gene [Cu/Zn-superoxide dismutase (SOD1) is a primary anti-oxidative enzyme that catalyzes the conversion of superoxide anion radicals into less reactive hydrogen peroxide and molecular oxygen. On the other hand, SOD1 is known to be an ALS-causative protein, and the aggregation of mutant SOD1 is a pathological hallmark in familial forms of ALS with mutations in the OD1 gene –16. AlthOD1 gene , 18, theOD1 gene . SOD1 isOD1 gene , ALS-linOD1 gene , 22 and OD1 gene . The redOD1 gene –27. ThioOD1 gene . NonetheD-value) and the effective acoustic thicknesses when the crystal oscillation decays using a flow-cell system. QCM-D monitors viscoelastic changes accompanying multilayer amyloid deposition [D-value of the sample adlayer on the sensor surface.Quartz crystal microbalance (QCM) techniques have been developed for obtaining, not only changes in mass but also the viscoelastic properties of molecules. Generally, QCM detects the mass of materials that had been adsorbed to the electrode surface as a frequency change, which is related to mass uptake via the Sauerbrey equation . Howeverposition , 31 and position . An alteIn this study, we report that an ALS-causing stable globular protein, SOD1, undergoes hydrogelation or aggregation accompanied by the formation of amyloid-like fibrils and that these conversions are pH-dependent. Monitoring of the immobilized SOD1 molecules with QCM-A permitted the different properties between water-rich extended molecules to be distinguished, under fibrillation and hydrogelation conditions (pH 3.0 −4.0) and water-poor compact molecules under precipitation conditions (pH 5.0).111 (Cys111-S-S-CH2CH2OH) was generously donated by Ube Industries Ltd. The fact that 2-ME only modified the side chain of Cys111, and not Cys6, was verified by MALDI–TOF MS (matrix-assisted laser-desorption ionization–time-of-flight MS) analysis [All chemicals used in this study were purchased either from Wako Pure Chemical Industries Ltd. or Sigma-Aldrich, unless specified otherwise, and were of the highest grade available. Recombinant wild-type human SOD1 (residues 1–153) modified by disulfide bonding with 2-mercaptoethanol (2-ME) at Cysanalysis . This 2-analysis , 34. GelSOD1 fibrillation was monitored by following the increase in the fluorescence intensity of thioflavin T (ThT). Two hundred μL of sample solutions containing various concentrations of SOD1, with or without DTT, EDTA and 20 μM ThT in buffers at various pH values in a 96-well plate were incubated at 37°C, and the ThT fluorescence intensity was recorded at 2 min intervals using a plate reader (Tecan Infinite M200 PRO) with excitation and emission wavelengths of 450 and 485 nm, respectively. The plate was shaken for 1 min at 200 rpm prior to each fluorescence reading.SOD1 hydrogels or SOD1 aggregates were absorbed on STEM100Cu elastic carbon coated grids , washed with water and then negatively stained with 2% Samarium (III) acetate hydrate. Images were obtained using an electron microscope .2ME-SOD1  was incubated overnight with 10 mM EDTA and 100 mM dithiothreitol (DTT) in a 50 mM acetate buffer (pH 3.0−5.0) or phosphate buffer (pH 6.0−8.0) at 37°C without shaking, which is a similar method for producing monomeric SOD1 .d = 40 mm, cone angle θ = 0.7 rad) equipped with a solvent trap filled with water to prevent evaporation. The SOD1 hydrogels were transferred to the stage and the frequency dependences of the complex moduli (Gʹ and Gʹʹ) were recorded under a constant shear strain of 2% maintained over the angular frequency range of 0.1 to 100 rad/s at 37°C. The amplitude dependences of the complex moduli were measured from 0.1% to 10% shear strain with an angular frequency of 20 rad/s at 37°C to investigate fragility of the hydrogels. When the network in the gel is broken by large amplitude strain, the complex modulus would change. For measurements of viscoelastic properties of the initial hydrogelation of SOD1 in a 50 mM an acetate buffer (pH 3.0–5.0) containing 10 mM EDTA and 100 mM DTT, rheological parameters, Gʹ and Gʹʹ, were monitored at 1 min intervals with an angular frequency of 20 rad/s at 37°C with 5% shear strain.Rheological measurements were carried out using a stress-controlled rheometer  with a cone-plate geometry  containing 10 mM EDTA and 200 mM DTT, followed by starting the measurement at 37°C with stirring  and the frequency parameters were recorded at 2 sec intervals. After monitoring, effective acoustic thickness, mass, D-value and shear viscosity were calculated.A 27 MHz QCM device, AFFINIX QN Pro , based on admittance (QCM-A) analysis  was usedReduction of the intrasubunit disulfide bond (Cys57-Cys146) and a loss of metals lead to the fibrillation of human SOD1 . Thus, tGʹ) and the loss moduli (Gʹʹ) of both hydrogels reached ~200 Pa and 20−30 Pa, respectively (Gʹ) response is superior to the viscous (Gʹʹ) one at the low frequency limit, Gʹ > Gʹʹ, the material is considered to be a solid. In the contrary case (Gʹʹ > Gʹ), the material is considered to be a liquid. Therefore, the SOD1 hydrogels are completely considered to be a solid. The properties of the SOD1 hydrogels are similar to those for a 0.25% agarose gel, showing ~300 Pa of Gʹ and ~10 Pa of Gʹʹ  were denatured in glass vials under fibrillation conditions in pH 3.0, 4.0 and 5.0 buffers. As shown in ectively . When tha of Gʹʹ . A sheara of Gʹʹ , which ia of Gʹʹ . These rFs  caused by the oscillation of a material adsorbed on the electrode. In addition, −2ΔFw/Fs is approximately equal to the energy dissipation value  obtained by the QCM-D method  containing 10 mM EDTA and 200 mM DTT for denaturation of SOD1. Most notably, in the case of pH 4.0, clear declines in ΔFw and ΔFs were observed after a 2 to 3 h treatment period and reached a steady-state level after a 15 h period (Fs) of immobilized proteins on the QCM-A sensor indicates the coupled water molecules, which could be detected even in aqueous solutions [Fw and ΔFs and a slight increase in ΔF2 were also observed in the case of pH 3.0. In contrast, almost no changes in ΔFw, ΔFs and ΔF2 were observed in the case of pH 5.0 until 15 h after the treatment. Changes in ΔFw, ΔFs and ΔF2 after denaturation for 15 h for the three experiments are indicated in D-value (ΔD)   and the lue (ΔD)  of the pTo exclude the effect of chemical immobilizing, the direct immobilized SOD1 was next used. Similar patterns of the frequency changes as in pKR is pH 4.25), which might affect hydrogen bonding with water molecules as well as the hydrophobicity of SOD1.As described above, hydrogel formation of SOD1 was strongly affected by the pH of the buffer. We also examined hydrogel formation where the pH is lower than 3.0 and pH 4.5. The SOD1 sample at pH 2  also formed hydrogels  and rapiSeveral reports have demonstrated that alterations in the ionic strength of the solution can influence the rate of formation of amyloid fibrils –40. We tFw and increase in ΔF2 , is 6.8 nm (between Gly130 of both subunits) and the maximum strand length is 3.4 nm between Ala1-Asp11 in the monomer . These d2, respectively, an increase from ~350 ng/cm2 for the native SOD1 that was immobilized on the sensor. It is estimated that ~1,000,000 water molecules could be coupled to one SOD1 monomer at pH 3.0. The denatured SOD1 adlayers containing such large amounts of water molecules appear to have the same viscosity as pure water  required a much longer time for fibrillation . AlthougOur findings are summarized in According to a general model pathway, “globular proteins first unfold and lose their secondary structure, which is the intermediate state, and some of them further form amyloid fibrils and subsequently self-assemble into hydrogels” . HoweverGʹ and Gʹʹ over time, are used because rheology is considered to be the most sensitive method for material characterization compared to chemical analyses [Gʹ and Gʹʹ for all solutions immediately started to increase −4.0, but with precipitates being formed at pH 5.0. The QCM-A analysis showed that the immobilized SOD1 on the sensor under the hydrogelation conditions at pH 3.0−4.0 resulted in an extension accompanied by the binding of large amounts of water molecules. In contrast, at pH 5.0, the denatured SOD1 hardly bound water molecules and remained compact. On the other hand, the addition of NaCl to the pH 4.0 sample caused a further water-rich intermediate, which resulted in the formation of an aggregation like emulsion but not a hydrogel. These results suggest that different denatured intermediate states of the protein before self-assembly play a major role in determining the characteristics of the resulting aggregates, such as fibrillar hydrogels, precipitates or aggregates like emulsions. An approach using the viscoelastic properties to monitor protein unfolding and water-protein interactions would be promising in terms of developing a further understanding of protein fibrils and the development of therapeutic strategies for the treatment of neurodegenerative diseases.S1 File(PDF)Click here for additional data file.S1 Fig(A) Conductance wave of the resonance frequency of the crystal oscillator. (B) Expected conductance waves and frequencies when hard materials (left panel) or soft materials (right panel) accumulated on the sensor.(TIF)Click here for additional data file.S2 Fig(A) Various concentrations of agarose gels dissolved by a microwave were transferred to the stage of a rheometer. After cooling at 10°C for 10 min, storage moduli (Gʹ) and loss moduli (Gʹʹ) of the agarose gels measured at 10°C with 0.1% shear strain. (B) Strain dependence of Gʹ and Gʹʹ of the agarose gels measured at 10°C with an angular frequency of 20 rad/s.(TIF)Click here for additional data file.S3 FigData are shown as the mean ± SEM from three independent experiments.(TIF)Click here for additional data file.S4 Fig(A) The effective acoustical thickness of some proteins on the sensor immobilized directly. (B) The shear viscosity of some proteins on the sensor immobilized directly.(TIF)Click here for additional data file.S5 Fig(A) Images of vial inversion tests for SOD1 solutions (20 mg/mL) at pH 0.88 to 5.0 after incubation at 37°C for 16 h under fibrillation conditions; upright vial bottle (upper panel) and inverted vial bottle (lower panel). (B) Fibrillation kinetics of SOD1 solutions (5 mg/mL) under fibrillation conditions in buffers at pH 0.88 to 5.0, as monitored by thioflavin T fluorescence. Representative kinetic data of three ThT assays are shown.(TIF)Click here for additional data file.S6 FigGʹ and Gʹʹ, were monitored at 1 min intervals with an angular frequency of 20 rad/s at 37°C with a 5% shear strain. Initial evolution of storage moduli (Gʹ) (A) and loss moduli (Gʹʹ) (B).Rheological parameters, (TIF)Click here for additional data file.


Streptomyces and Brachybacterium species were evaluated. Optimum bioflocculant yields (g/L) and flocculation activities (%) were observed for the following preferred nutritional sources: glucose , (NH4)2NO3  and CaSO4·H2O . A Plackett-Burman design revealed the critical fermentation media components. The concentrations of these components were optimized  through a central composite design with optimum bioflocculant yield of 3.02 g/L and flocculation activity of 63.7%. The regression coefficient (R2 = 0.6569) indicates a weak estimation of the model’s adequacy and a high lack-of-fit value (34.1%). Lack of synergy in the consortium may have been responsible for the model inadequacy observed. FTIR spectrometry showed the bioflocculant to be a heteropolysaccharide, while SEM imaging revealed an amorphous loosely arranged fluffy structure with interstial spacing of less than 1 µm. The biofloculant production potential of a consortium of  Biopolymeric materials, of extracellular or intracellular origin, synthesized by some species of bacteria, fungi and algae have been variously documented to mediate flocculation of suspended particles in liquid media ,2,3,4. TNeurodegenerative diseases such as Alzheimer’s have been associated with polyaluminum chlorides , while tAppreciable flocculation activities has been reported for bioflocculants produced by several prokaryotes, fungi and a few algae ,13,14 hoBesides bio-prospecting for novel bioflocculant-producing bacteria, strategies employed for yield optimization of microbial products include mutational analysis and manipulation of nutritional and fermentation conditions . Mixed cBrachybacterium sp. UFH and Streptomyces sp. Gansen produced bioflocculants characterized as composed of uronic acids, polysaccharide, and proteins, among other components. The bioflocculants produced by the respective actinobacteria were stable to pH extremes and high temperature. Besides the optimization of yield through manipulation of fermentation conditions for the axenic cultures, further optimization process was attempted through evaluation of Brachybacterium sp. and Streptomyces sp. as a mixed culture. The critical fermentation media components were determined with the Plackett-Burman (PB) experimental model, while a central composite design (CCD) was used to optimize the identified critical media components. Application of PB and CCD was necessitated by the dearth of information on media optimization for mixed culture fermentation. The bioflocculant produced was purified and characterized.In our previous studies, axenic cultures of Streptomyces sp. and Brachybacterium sp. consortium to optimally utilize glucose, (NH4)2NO3 and CaSO4·H2O respectively (4)2NO3], 49% and 1.96 ± 0.21 g/L (urea), 38% and 1.99 ± 0.56 g/L respectively (4·H2O and MgCl2 (4)2NO3] and CaSO4·H2O were the preferred nutritional sources as they respectively yielded the optimal flocculation activity, the difference in flocculation activities achieved with other nutritional components, were not statistically significant (p ≤ 0.05). The different carbon, nitrogen and cation sources evaluated for optimal utilization for bioflocculant production showed ectively . The floectively . The catnd MgCl2 . AlthougBrachybacterium sp. UFH and Streptomyces sp. Gansen seems to have acted in an antagonistic manner thus leading to the decline of bioflocculant yield and flocculation activity, respectively. The actinobacterial species responsible for the antagonistic effect is not known however, the decreased bioflocculant yield was taken as an indication of antagonism. Nonetheless, the consortium produced bioflocculant in an amount lower than the respective axenic cultures as noted by the yields and flocculation activity. A similar trend was observed with the assessed nitrogen and cation sources. Hence, yield optimization through mixed culture fermentation is achieved only when the respective culture acts in synergy . HoweverThe utilization of various nutrient sources for the production of microbial secondary metabolites have been reported for axenic cultures ,15,25,264)2NO3, CaSO4·H2O, K2HPO4 and KH2PO4 were evaluated for their respective contributions towards bioflocculant production with an experimental outlay (p ≥ 0.05). Flocculation activities of 57% and 56% were recorded at experimental trials No. 4, 6 and 11 as the optimum (4)2NO3], 0.5 (CaSO4·H2O), 5.0 (K2HPO4) and 2 (KH2PO4) for trail No. 4, 12.5 (glucose), 1 [NH4)2NO3], 0.3 (CaSO4·H2O), 5.0 (K2HPO4) and 2.5 (KH2PO4) for trials No. 6 and 10 (glucose), and 1 [NH4)2NO3], 1 (CaSO4·H2O), 5.0 (K2HPO4) and 2 (KH2PO4) for trial No. 11, respectively. In addition, the regression analysis indicates that glucose, (NH4)2NO3 and CaSO4·H2O had positive effects on bioflocculant production, unlike K2HPO4 and KH2PO4 (2); 0.5 (glucose), 0.4 [(NH4)2NO3] and 0.1 (CaSO4·H2O) shown by the respective nutritional sources indicated very weak impact on the production of bioflocculant by the consortium 2NO3 and CaSO4·H2O, although their significance with respect to bioflocculant production with the consortium was low, with only glucose barely surpassing 50% while CaSO4·H2O was estimated to have about an 11% influence. Dipotassium hydrogen phosphate and potassium dihydrogen phosphate showed a negative input, respectively, on the production of bioflocculant, hence their input may be deemed as insignificant. Nonetheless, besides bioflocculant production these salts may have served in a pH buffering function thus maintaining the physiological pH of the culture balanced. The identification of critical media components serves to reduce the cost of fermentation if industrial application is envisaged. The critical media components identified through the Plackett-Burman design model were glucose, (NH4)2NO3 and CaSO4·H2O were next optimized in a 3-factor-5-level central composite design 2NO3 and CaSO4·H2O, respectively, following the twenty experimental trials shown in the 3-factor-5-level CCD matrix. The flocculation activity and bioflocculant yield achieved at this media components optimum were 63.7% and 3.02 g/L, respectively. The respective proportion of critical media with the highest flocculation activities were 16.0 g/L, 0.5 g/L and 1.6 g/L of glucose, (NH2 = 0.6569), implied a 65.69% variability with respect to enhancing bioflocculant production as earmarked by the flocculation activity shown by Brachybacterium sp. and Streptomyces sp. consortium. Following analysis of variance, the response surface was fitted to a second order model . The rel2 = 0.341) which was not statistically significant (p ≤ 0.000025). Hence, there is an indication of the model adequacy for the prediction of enhanced flocculation activity following the assay conditions 2NO3 and CaSO4·H2O to the production of bioflocculant were 1.8%, 91.3% and 28.9% respectively, as indicated by the linear model (4)2NO3 and CaSO4·H2O showed positive contribution to bioflocculant yield while glucose did not. The negative regression coefficients shown by glucose following the quadratic polynomial model is an indication of the low impact shown by this carbon source towards enhancing bioflocculant yield during fermentation by the consortium. The interaction between glucose, (NH4)2NO3 and CaSO4·H2O showed that (NH4)2NO3 and CaSO4·H2O was significant while the rest was not, as their coefficients of estimate were negative 2NO3 flocculation activities increased (4)2NO3 apparently showed no increase in flocculation activity at any level (Brachybacterium sp. and Streptomyces sp. were: 16.0 g/L (glucose), 0.5 g/L [(NH4)2NO3] and 1.2 g/L (CaSO4·H2O), respectively. The maximum flocculation activity and bioflocculant yield achieved were 63.7% and 3.02 g/L, respectively. The three dimension surface response plot  showing ncreased A. Likewincreased C while tny level B. Conseq4)2NO3 and no clear reason can be adduced to this observation as carbon sources are known to be the most relevant factors for microbial growth. On the other hand, it may be that nitrogen sources were more important in bioflocculant production. Microbial secondary metabolites including poly-γ-glutamic acid from Bacillussubtilis RKY3 [Halomonas sp. V3a’ [The feasibility of optimizing critical media components is grim if cultures are unable to grow effectively. However, since we did not ascertain the survival of the respective axenic culture in the consortium then, it will be prudent not state that growth was poor particularly as it has been shown that bioflocculant production occurs at the exponential phase of bacterial growth ,30. The lis RKY3 , bioflocsp. V3a’ , biosurfsp. V3a’  and in tsp. V3a’  have bee−1), characteristic of hydroxyl groups from polymeric, dimeric and monomeric OH groups. Similarly, peaks from 2958.70 to 2854.39 cm−1 correspond to weak C–H stretching bands from methylene groups, and those from 1654.77 to 1539.01 cm−1 are indicative of the presence of aromatic rings [−1 and 1242.18 to 1047.30 cm−1 shown were typical of phenol and tertiary alcohol OH bend, indicative of the presence of carboxylic groups, carboxylate ions, aromatic ring stretch and C–O and C–O–C from polysaccharides [Electron micrographic imaging of the purified bioflocculant showed loosely packed fluffy materials with irregular arrangement patterns . The intic rings ,35. Furtcharides . The loose amorphous fluffy nature of the bioflocculant is a marked variation from the clump-like nature shown by bioflocculants with high flocculation activity . SimilarBrachybacterium sp. UFH (accession number HQ537131) and Streptomyces sp. Gansen (accession number HQ537129), preserved at −80 °C as part of the culture collection of the Applied and Environmental Microbiology Research Group (AEMREG), University of Fort Hare, South Africa were reactivated by inoculating 20 µL of the glycerol stock into a sterile 5 mL sterile broth composed of 3 g beef extract, 10 g tryptone and 5 g NaCl (per liter), respectively, and incubated overnight at 28 °C. Cultures of Brachybacterium sp. UFH and Streptomyces sp. Gansen were adjusted to cell densities of about 1.5 × 108 cfu/mL and aliquots of 2 mL were inoculated into 200 mL of sterile basal salt media composed of the following (g/L): glucose, 10; tryptone, 1; K2HPO4, 5; KH2PO4, 2 and MgSO4·7H2O, 0.3. The fermentation medium was adjusted to pH 7 and incubated at a temperature of 30 °C with an agitation speed of 160 rpm for a period of 72 h. The broth, after the incubation period, was centrifuged at 3,000 rpm for 30 min at 15 °C and the cell-free supernatant was assessed for flocculation activity. Fructose, sucrose, lactose, maltose and starch respectively served as sole carbon sources, while the sole nitrogen and cation sources evaluated included urea, ammonium sulphate, ammonium nitrate, ammonium chloride, peptone, monovalent salts (KCl and NaCl), divalent salts  and trivalent salts (FeCl3), respectivelyThe activated actinobacteria species, 2 and 0.2 mL of cell free broth (bioflocculant rich broth) were added to 10 mL of kaolin suspension (4.0 g/L) in a test tube. The mixture was vortexed using a vortex mixer  for 30 s and kept still for 5 min, after which 2 mL of the upper layer was carefully withdrawn and its optical density (OD) read spectrophotometrically  at 550 nm wavelength. Control included repeating same process however, the bioflocculant broth was replaced with sterile (un-inoculated) fermentation medium [550  of the control and sample, respectively.About 0.3 mL of 1% CaCln medium . All ass4·H2O and (NH4)2NO3, K2HPO4 and KH2PO4 which were investigated at two levels (concentrations) of each variable, ‘‘high” and ‘‘low” were used and was designated as +1 and −1 respectively (p < 0.05) effect on flocculation activity, and these components were evaluated in further optimization experiments. NCSS 2007 , was used to design and developed the PB experimental design based on the following first-order model:Critical media components for the production of bioflocculant by the mixed culture were assessed using the Plackett-Burman (PB) design in an “n” variable screening of n + 1 experiments . The carectively . All exp4·H2O and (NH4)2NO3 were fitted into the model using the 3-factor-5-level CCD [Media components identified by the PB design as critical for bioflocculant production were optimized through the response surface methodology (RSM). A central composite design (CCD) model was generated and critical media components; glucose, CaSOevel CCD . ExperimThe fermentation broth was centrifuged  and cell pellets separated from the supernatant by decantation. The supernatant was mixed with ice cold ethanol (95%), at volume to volume ratio of 1:4 and kept at 4 °C in a cold cabinet for 16 h. The ethanol and cell free broth mixture was centrifuged  and the residue redissolved in distilled water at a ratio of 1:4 (v/v). The procedure was successively repeated twice and the purified bioflocculant was lyophilized and vacuum dried . The lyo−1. Purified bioflocculant was placed on carbon coated stub and gold coated in a gold coating chamber, using Eiko IB.3 ION coater. Scanning electron microscopic (SEM) image of the gold coated bioflocculant was obtained using JEOL JSM-6390LV FEI XL30  scan electron microscope. Functional groups present in the bioflocculant were determined using a Fourier transform infrared (FT-IR) spectrophotometer  over a wavenumber range of 4000 to 500 cmBrachybacterium sp. UFH and Streptomyces sp. Gansen produced bioflocculant with low flocculation activity and in low yield when compared to the respective axenic culture. The application of response surface design marginally improved the yield however, the model was not adequate as the antagonistic effect of the culture metabolites impeded effective synthesis of bioflocculant. Although mixed culture is an effective tool in optimization of desired effects, synergy is essential for the desired effects to be achieved. In conclusion, the consortium of


These studies adequately prove that trehalose at an appropriate dose would be potentially useful for PD treatment.Fibrillar accumulation of A53T mutant α-synuclein (A53T-AS) in Lewy bodies is a symptom of Parkinsonism. Inhibitions of the overexpression and fibrillar aggregation of α-synuclein (AS) in vivo could be a promising strategy for treating Parkinson’s disease (PD). In this study, at concentrations lower than 1 mM, trehalose decreased the A53T-AS expression level in transduced PC12 cells. Although H Parkinson’s disease (PD) is the second most common disease among nine inherited devastating neurological disorders. Clinically, PD is a slowly progressive movement disorder characterized by tremor, muscle rigidity, imbalance, and bradykinesia. These symptoms are due to the progressive loss of dopaminergic neurons from the substantia nigra, which is a small area in the midbrain. Moreover, some surviving nigral dopaminergic neurons contain cytosolic filamentous inclusions, known as Lewy bodies and Lewy neurites .2O2, caused mitochondrial dysfunction and cell death [α-Synuclein (AS) is an intrinsically disordered protein ,3,4 thatll death ,13, and ll death .Currently, PD is considered to be an “environmental” disease . The posTrehalose is a non-reduced disaccharide  that hasThe present work aimed to investigate the effect of trehalose at concentrations lower than 10 mM on the overexpression of A53T-AS, and the neuroprotection against the protein aggregation by using PC12 cells as model cells that induce A53T-AS expression. This problem has been receiving limited attention in the past research. We also sought to demonstrate trehalose as a neuroprotective agent for the treatment of PD.To reveal the effect of trehalose on A53T-AS overexpression, we employed the rat pheochromocytoma cell line PC12 expressing A53T-AS. 2O2 and 2 µM Al (III), A53T-AS expression level was significantly higher than that with 5 mM trehalose. To study the effect of trehalose on the Al (III)-induced expression of A53T-AS, transduced PC12 cells were incubated with 2 µM Al (III) along with 5 mM trehalose. Compared with those samples solely containing Al (III) or trehalose, A53T-AS expression was apparently inhibited by Al (III) and trehalose  or H2O2 along with 5 mM trehalose, the A53T-AS expression was obviously inhibited, and the MMP level was increased, compared with the sample with Al (III) or H2O2 alone. rehalose . To furtecreased . To furt2O2 and Al (III) was studied by 3--2,5-diphenyltetrazolium bromide (MTT) assay  significantly attenuated the cell viability to 68%  assay . Figure y to 63% b. Compary to 68% c. Furthe2O2 is one of the ROS that induced the intracellular overexpression of A53T-AS. Considering the effect of trehalose on the fibrillation of A53T-AS in the previous work [Understanding the impact mechanism of trehalose on A53T-AS overexpression and neurotoxicity on a cellular basis is a paramount issue for the development of trehalose for clinical applications. AS fibrillation plays an important role in neurodegeneration in Parkinson’s disease; in particular, the oligomeric fibrillary aggregates of AS are toxic to neurons . Combinious work , trehaloous work . That thous work , possibl2O2 and Al (III) can accelerate the formation of A53T-AS toxic species, and the trehalose concentration (5 mM) itself is also toxic, the complex of Al (III) with trehalose could hinder the toxicity of Al (III) in cells by preventing A53T-AS conformational transition. However, when low trehalose concentrations were co-incubated with Al (III), the effect was not sufficiently strong to ameliorate Al (III) toxicity in cells.Sugar transportation onto cells is inhibited by several metals, such as nickel, cobalt, aluminum, and cadmium ,24. The 2O2-induced cell death by inhibiting the Al (III)- or H2O2-induced intracellular A53T-AS overexpression, and decreasing ROS in cells  and He damage . Thus, tTM Lentiviral Expression System, Invitrogen), as previously described [The PC12 cell is a cell line derived from a pheochromocytoma of the rat adrenal medulla and was purchased from Shanghai Institutes for Biological Sciences, CAS, Shanghai, China. PC12 cells that can overexpress the wild-type AS were prepared as described in our previous reports . Brieflyescribed . After Al-glutamine (3.6 mM), and 1% penicillin/streptomycin antibiotics under 5% CO2 atmosphere at 37 °C. Cells were harvested from flasks and plated in six-well polystyrene plates  with 5 × 105 cells in 2.5 mL medium per well, and then incubated at 37 °C for 24 h to attach the plate. Two µM Al (III), 100 µM H2O2, and 0.2, 0.5, 1, 5 and 10 mM trehalose were then added individually to those samples. The selection of concentrations used for trehalose and H2O2 was based on our previous study [g for 10 min, supernatants were collected. Protein concentrations in supernatants were determined by BCA method . Before electrophoresis, cell lysates in supernatants were further diluted with SDS loading buffer  and heated at 100 °C for 5 min. A total of 30 μg of lysate was separated by SDS-PAGE, and transferred to polyvinylidene difluoride (PVDF) membranes . The transformation to PVDF membrane was then blocked in TBST  and probed by the rabbit anti-human AS primary antibody . The protein was detected using enhanced chemiluminescence reagents , according to the manufacturer’s instruction. The intensity of each band was estimated by densitometric quantification using the Image Lab version 5.2.1 software (Bio-Rad). Data were collected from three independent experiments.The transduced PC12 cells expressing A53T-AS were grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 6.7% horse serum, 3.3% fetal bovine serum (FBS), 1% us study . The saml-glutamine (3.6 mM), and 1% penicillin/streptomycin antibiotics under 5% CO2 atmosphere at 37 °C. Cells were harvested from flasks and seeded on 0.01% poly-l-lysine-coated  glass slides in 12-well polystyrene plates , with 5 × 104 cells in 1 mL medium per well, and incubated at 37 °C for 24 h to attach the plate. Then, the pre-incubated medium in each well was aspirated carefully, and 1 mL fresh medium , and 1% penicillin/streptomycin antibiotics) with 0, 1, and 10 mM trehalose were added individually to each cell well. Cells were incubated for an additional 48 h at 37 °C, and then stained with 10 µM rhodamine 123 (Rh123)  for 30 min. Cells were fixed in 4% (w/v) paraformaldehyde in phosphate buffer saline (PBS) for 20 min. Upon washing with PBS, cells were permeated with 0.1% Triton X-100 in PBS and blocked with 3% goat serum (Songon Biotech) in PBS for 30 min at room temperature, subsequently incubated with rabbit anti-human AS primary antibody  for 1 h, and then incubated with a secondary antibody  for 1 h. DAPI  and TRITC-conjugated phalloidin  was used to label nuclei and actin filament, respectively. After a final washing with PBS, coverslips were mounted in the antifade mounting medium  and a laser scanning confocal microscope  was applied to capture images.The transduced PC12 cells expressing A53T-AS were grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 6.7% horse serum, 3.3% fetal bovine serum (FBS), 1% l-glutamine (3.6 mM), and 1% penicillin/streptomycin antibiotics under 5% CO2 atmosphere at 37 °C. Cells were harvested from flasks and plated in 96-well polystyrene plates with 5.5 × 103 cells in 100 µL medium per well. Cells were incubated at 37 °C for 24 h to attach the plates. Then, the pre-incubated medium in each well was aspirated carefully, and 100 µL fresh medium , and 1% penicillin/streptomycin antibiotics) with 0.2, 0.5, 1, 2 and 5 mM trehalose or 10 and 100 µM H2O2 or 2 and 10 µM Al (III) or their mixtures were added individually to each cell well. The same volume of medium was added to the control well (only PC12 cells present). Cells were then incubated for additional 48 h at 37 °C. At the end of incubation, 20 µL fresh DMEM with 3.3 mg/mL thiazolyl blue tetrazolium bromide was added to each well and incubated for additional 4 h at 37 °C for the formation of formazan crystals. The formed crystals were then dissolved by adding 100 µL DMSO in each well and shaking for 20 min at room temperature. The absorbance was measured at 490 nm by Bio-Rad multi-well assay plate reader, and averaged by five replicated wells for each sample and control. The cell viability was calculated by the ratio of the absorbance of studied groups to that of the control group. Data were collected from three independent experiments. Standard deviations were analyzed by one-way ANOVA packaged in Origin 7.5 software .Transduced PC12 cells were grown in DMEM containing 6.7% horse serum, 3.3% FBS, 1%