Argiope argentata, which lacks several well conserved residues involved in the dimerization of other NTs. We solve its structure at low pH revealing an antiparallel dimer of two five-α-helix bundles, which contrasts with a previously determined Nephila antipodiana TuSp NT monomer structure. Further, we study a set of mutants and find that the residues participating in the protonation events during dimerization are different from MaSp and MiSp NT. Charge reversal of one of these residues (R117 in TuSp) results in significantly altered electrostatic interactions between monomer subunits. Altogether, the structure and mutant studies suggest that TuSp NT monomers assemble by elimination of intramolecular repulsive charge interactions, which could lead to slight tilting of α-helices.The spidroin N-terminal domain (NT) is responsible for high solubility and pH-dependent assembly of spider silk proteins during storage and fiber formation, respectively. It forms a monomeric five-helix bundle at neutral pH and dimerizes at lowered pH, thereby firmly interconnecting the spidroins. Mechanistic studies with the NTs from major ampullate, minor ampullate, and flagelliform spidroins  have shown that the pH dependency is conserved between different silk types, although the residues that mediate this process can differ. Here we study the tubuliform spidroin (TuSp) NT from  Orb weaving spiders stand out among silk producers found in nature as they can produce up to seven different types of silk, each tailored for a specific function. The main components of the spider silk threads are large proteins called spidroins. Major ampullate spidroin (MaSp) is the main component of dragline silk, the strongest and toughest of all spider silk types . Spider 2 pressure) and increasing shear forces cause them to assemble and form solid threads  and the C-terminal domain (CT) , whereasEuprosthenops australis MaSp NT dimer  to characterize the TuSp NT conformation at pH > 7 and to determine its structure at pH 5.5. Next, several methods including NMR, size exclusion chromatography (SEC), and circular dichroism (CD) are used on the wild type (wt) protein as well as site-directed mutants to investigate the dimerization mechanism of TuSp NT.Considering the additional mechanistic insights gained from the recent studies of MaSp, MiSp and FlSp NT and the contrasting structural data of TuSp NT, we decided to re-investigate its structure and possible dimerization mechanism using a protein construct that comprises the complete conserved domain sequence from A. argentata TuSp 1 NT sequence and pET-28a-HisNT containing TuSp NTE37QE82Q and TuSp NTE37QE82QE85Q were ordered from BioCat GmbH . For expression of TuSp NT, TuSp NTA70E, and TuSp NTA70R the pET-32a vector was used. TuSp NTD40RR63D, TuSp NTE37QE82Q, TuSp NTE37QE82QE85Q, and TuSp NTE37QE82QE85QD130N were expressed by using the pET-28a vector . At first, two PCR reactions were carried out in parallel with forward and reverse primers in separate tubes. After PCR, the two products were combined in a 1:1 ratio and another thermocycle was run. PCR products were digested with DpnI, and purity of plasmids was checked on 1% agarose gel. The plasmids were used to transform chemically competent E. coli XL1-Blue cells by heat shock, which was followed by plasmid extraction and sequence verification.pET-32a-HisTrxHisNT plasmid containing wt E. coli BL21 (DE3) cells. Overnight cultures were inoculated at a ratio of 1:100 into LB medium containing 50 μg/ml kanamycin (pET-28a plasmids) or 100 μg/ml ampicillin (pET-32a plasmids) and cells were further grown at 37°C and 180 rpm to an OD600 of 0.6. Expression was induced by addition of isopropyl β-D-1-thiogalactopyranoside (IPTG) to a final concentration of 0.5 mM and cultures were further incubated overnight at 25°C. The cells were harvested by centrifugation at 7000 g for 10 min and stored at −20°C.The plasmids were used for heat shock transformation of chemically competent Cells were thawed and resuspended in the immobilized metal-affinity chromatography (IMAC) loading buffer . The cells were sonicated, and the lysate was cleared by centrifugation at 20000 g for 40 min. The supernatant was then loaded on a 2 × 5 ml HisTrap HP column (Cytiva). Unbound proteins were washed off with 5 column volumes of loading buffer. Bound proteins were eluted in 2 ml fractions using 500 mM imidazole, 20 mM Tris-HCl, 8 M urea, 300 mM NaCl, 1 mM DTT, pH 8.0. The absorbance at 280 nm was measured for each fraction and protein-containing fractions were pooled and dialyzed against 20 mM Tris-HCl, 300 mM NaCl, 1 mM EDTA, 1 mM DTT, pH 8.0 at 4°C. Complete refolding of the protein was verified by NMR. Fusion proteins were cleaved with tobacco etch virus (TEV) protease  during dialysis against TEV reaction buffer  at 4°C. After the cleavage, sample was loaded on the HisTrap HP column to remove the cleaved fusion tag and unbound target protein was collected. Protein was further purified by gel filtration with a HiLoad 16/600 Superdex 75 pg size exclusion column (Cytiva). The concentration of protein was determined by using the Pierce Bicinchoninic Acid (BCA) protein assay kit  according to the manufacturer’s recommendations. The correct size of protein was confirmed by SDS-PAGE using 4–12% Bis-Tris polyacrylamide gel , stained with Coomassie brilliant blue dye.15N- and 13C, 15N-labeled samples the same procedure was used except that M9 minimal medium containing 15NH4Cl and 13C-glucose as the sole sources of nitrogen and glucose, respectively, was used.For production of 3 (NaOAc-d3), pH 5.5, 20 mM NaCl, 0.03% (w/v) NaN3, 5% D2O (v/v) buffer. NMR spectra were acquired at 298 K on a Bruker Avance Neo 600 MHz spectrometer equipped with a TCI z-gradient cryoprobe. For backbone assignment, 3D HNCA, 3D HNCO, 3D CBCA(CO)HN, and 3D HN(CA)CO spectra were acquired. For side chain assignment and structure determination, 3D -nuclear Overhauser spectroscopy (NOESY)-15N-heteronuclear single-quantum correlation (HSQC), 3D -NOESY-13C -HSQC, 3D -NOESY-13C (aromatic)-HSQC spectra were acquired with a mixing time of 80 ms. Chemical shifts were referenced internally to the residual water signal at 4.77 ppm relative to 4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS).The protein sample used for NMR structure determination was concentrated to approximately 1 mM in 20 mM sodium acetate-d15N-1H HSQC NMR spectra, 15N-labeled samples were prepared in either 20 mM NaOAc-d3, 20 mM NaCl, pH 5.5 or 20 mM sodium phosphate (NaPi), 300 mM NaCl, pH 7.2 buffers. The spectra were acquired using either Bruker Avance III HD 800 MHz spectrometer equipped with an TXI z-gradient room temperature probe or Bruker Avance Neo 600 MHz spectrometer equipped with a TCI z-gradient cryoprobe.For recording of 2D1H-detected 15N relaxation experiments. The relaxation data were recorded on the Bruker Avance III HD 800 MHz spectrometer equipped with an TXI z-gradient room temperature probe. Spectra were processed in TopSpin 4.1.1 and signal intensities were measured with CARA 1.8.4.2  and seven T2 (10–250 ms) delays were used for the recording of the decay curves. Protein rotational correlation time (τc) was calculated using the equationN is the 15N resonance frequency (in Hz), T1 is average longitudinal and T2 average transverse relaxation time value of rigid protein regions.The molecular weight of TuSp NT at pH 5.5 was estimated using 2D  1.8.4.2 . Residueax 4.0.3 . Seven TN6T1T2−7, where νAll acquired spectra were processed using TopSpin 4.1.1 software. The assignment of protein backbone and side chain chemical shifts was performed with CARA 1.8.4.2 . The peaSize exclusion chromatography on the purified TuSp NT and its variants was performed using an Äkta Purifier 10 system. 50 µl of purified protein at a concentration of 3 mg/ml were loaded onto a Superdex 75 Increase 10/300 GL size exclusion column (Cytiva) previously equilibrated in the relevant buffer . The molecular weight calculation was performed according to the calibration found in the column manual, where conalbumin (75 kDa) was measured to elute at 8.7 ml, ovalbumin (44 kDa) at 9.65 ml, carbonic anhydrase (29 kDa) at 11.2 ml, ribonuclease (13.7 kDa) at 13.3 ml and aprotinin (6.5 kDa) at 16.1 ml.CD experiments were performed on a Jasco J-1500 CD spectrometer using 300 µl cuvettes with 1 mm path length. All measurements were acquired in 20 mM sodium phosphate buffer at pH 8.0 or pH 5.5 at 10 µM protein concentration. Spectra were recorded from 260 to 185 nm, first at 25°C, then after heating for 15 min at 95°C and finally after cooling down to 25°C. 5 scans were recorded for each temperature to calculate an average spectrum. For temperature melting curves, the CD intensity at 222 nm was monitored between 25 and 95°C with 1°C/min increase.A. argentata TuSp NT with N-terminal His-Trx fusion tag was expressed as insoluble inclusion bodies in E. coli BL21 (DE3). Following solubilization in 8 M urea, the protein was purified using IMAC and refolded by dialysis against Tris buffer at pH 8 and 4°C. The His-Trx solubility tag was subsequently cleaved with TEV protease. The cleaved protein was once again passed through an IMAC column to remove the tag and further purified by gel filtration in presence of 300 mM NaCl to isolate monomeric TuSp NT.15N-1H HSQC NMR spectroscopy. A high-quality spectrum was obtained only at low pH conditions corresponding to the spider’s spinning duct , as indicated by ∼130 well separated peaks with similar intensities . Secondly, the molecular dipole of each subunit important for the initial association of monomers was disrupted by charge reversal of the residues D40 and R63 (mutant TuSp NTD40RR63D). TuSp NTA70E and TuSp NTD40RR63D variants could be produced in sufficient quantities for NMR experiments using the same protocol as for the wt protein, whereas TuSpA70R could not be properly refolded. The inability to refold TuSp NTA70R is likely associated with formation of a new electrostatic interaction that is incompatible with the correct fold of the protein.As described above, at pH > 7 and in presence of high salt concentrations  TuSp NT showed a reduced number of HSQC NMR signals. To stabilize its monomeric conformation, three mutants were prepared by introducing single or double-site mutations, which prevent dimerization of MaSp NT . FirstlyA70E and TuSp NTD40RR63D at both, pH 7.2 and pH 5.5, were similar to the spectrum of wt TuSp NT at pH 7.2 suggesting that dimerization was prevented. However, for TuSp NTA70E at pH 5.5 and for TuSp NTD40RR63D at both pH values the spectra also showed peak broadening implying that the monomeric conformation was not fully stabilized , which matches well with the expected molecular mass of the TuSp NT dimer (28 kDa). The backbone assignment was performed using standard triple-resonance 3D experiments  that become protonated during MaSp NT dimerization, only E84 is conserved in TuSp NT (as E82). This residue shows a similar side chain orientation in the TuSp NT dimer, pointing towards D40 of the same subunit, with which it forms a characteristic handshake interaction in the MaSp NT dimer structure . Howevernformers . Thus, tnformers .A70E . The monomeric mutants TuSp NTA70E and TuSp NTD40RR63D were also included as controls.Unlike MaSp NT, the TuSp NT sequence does not contain the W10 residue, which prevents monitoring of the monomer-to-dimer transition by tryptophan fluorescence . Hence, Comparison of HSQC spectra of TuSp NT at pH 7.2 and 5.5 with the corresponding spectra of the three mutants indicated high structural similarity at pH 5.5 (corresponding to dimeric state) but less similar conformations at pH 7.2 . HoweverA70E at pH 8 was 11.0 kDa. As its HSQC spectrum showed no signs of partial unfolding, the reduced apparent molecular weight could suggest that this mutation indeed stabilizes the monomeric state. However, at low pH TuSp NTA70E eluted with an apparent molecular weight of 22.8. kDa indicating that it can still dimerize. TuSp NTD40RR63D eluted with apparent molecular weights of 18.0 (pH 8) and 21.7 kDa (pH 5.5) suggesting an intermediate state at both pHs, which is in line with presence of both species and with data from HSQC NMR experiments. The three acidic residue mutants TuSp NTE37QE82Q, TuSp NTE37QE82QE85Q, TuSp NTE37QE82QE85QD130N displayed elution volumes consistent with an intermediate state at pH 8 and dimer state at pH 5.5. Thus, all three mutants kept responsiveness to pH suggesting that we failed to create a constitutive dimer.SEC was performed in presence of 300 mM NaCl at pH 8 and 20 mM NaCl at pH 5.5. The experiments with TuSp NT showed that it migrates with an apparent molecular weight of 13.1 kDa at pH 8 and of 26.3 kDa at pH 5.5 . These vm) were determined from the half-denaturation points between the native and unfolded states. Dimerization of MaSp NT was shown to increase protein stability through neutralization of intramolecular repelling charge clusters  than reported for wt E. australis MaSp NT   at high pH and are converted into dimeric conformation at pH 5.5. CD experiments showed that thermal stability of the mutants is higher at pH 8, however, not approaching the stability of TuSp NT at pH 5.5.We performed structural studies and dimerization analysis of TuSp NT from variants , failed N. antipodiana shows an atypical four-helix arrangement that is distinct from the five-helix NT structures, raising questions about the fold conservation. Despite high sequence similarity (66% identity), our herein determined TuSp NT structure from A. argentata reveals a five-helix composition, which is highly similar to the structures of MaSp, MiSp, AcSp, and FlSp NTs. Superposition of the two TuSp NT structures shows a different organization of the first three helices  in presence of 100 mM DPC, whereas we employed acidic conditions . Accordingly, the N. antipodiana structure at pH 7 shows a monomer, whereas the A. argentata TuSp NT at pH 5.5 is a dimer. The use of a shorter construct and presence of 100 mM DPC, which according to the authors was necessary to avoid aggregation, raises concerns about the relevance of the N. antipodiana structure. Our study shows that the structured part of TuSp NT begins already at residue 10, both for the wt protein at pH 5.5  is structurally preserved, whereas E79 and E119 are replaced by D77 and R117, respectively, and form an intermolecular salt bridge in the dimer structure. These substitutions alter the electrostatic surface potential of each monomer subunit, making the positively charged pole more extensive (i.e. spread out) than in MaSp NT . In the  MaSp NT , which c MaSp NT . Thus, tsequence  could abE37QE82Q showed that some other residue is additionally protonated at low pH. The only other well conserved glutamate residue among TuSp NTs is E85, which in some conformers of our structure is spatially close to D130 of the same subunit and the charge clash could prevent it from assuming a dimer-compatible conformation. However, preparation and characterization of the triple and quadruple mutants TuSp NTE37QE82QE85Q and TuSpE37QE82QE85QD130N gave similar results as for TuSp NTE37QE82Q. This result does not rule out the involvement of at least some of the mutated residues in the observed pH sensitivity, because the studied combinations may bear disruptive mutations affecting the pKa of other residues that participate in the protonation events of the wt protein.Since E82 shows a similar side chain orientation to E84 in MaSp NT and additionally has E37 located in close proximity, we hypothesized that neutralization of these two residues may be important for establishing the characteristic handshake interaction between E82 and D40 as seen in the MaSp NT dimer. Although E37 is not conserved in most other spidroins, it is highly conserved between TuSp NTs from different species . HoweverIn summary, our obtained results clearly indicate that the mechanism of pH-dependent dimerization is different for TuSp NT than it is for MaSp and MiSp NT. We show that in contrast to previous findings TuSp NT has the same five-helix fold of other NTs and forms a stable dimer at low pH. However, its unique amino acid sequence does not allow full stabilization of the monomer conformation at the conditions, where MaSp NT forms stable monomers (pH 7.2 in presence of 300 mM NaCl). This may be linked to the lack of a well-defined spidroin storage sac as well as different physiological conditions  in the tubuliform glands, which remain to be characterized. Furthermore, the pH dependent stable dimer formation that takes place in the silk duct upon fiber formation involves a very different set of amino acid residues as compared to MaSp and MiSp NT. Further research is needed to identify the exact amino acid residues that become protonated in the TuSp NT dimer at low pH.


Single-phase samples are prepared for 0 ≤ x ≤ 3 and show a complex structural evolution from P2/c to C2 space groupswith an increase in x. For 1.0 ≲ x ≲ 2.4, average structures determined by X-ray and neutronpowder diffraction show metrically orthorhombic unit cells, but HAADF-STEMimaging reveals this is caused by microstructural effects due to intergrowthsof the Ba- and Sr-rich structure types. Variable-temperature powderdiffraction studies suggest that 0 ≲ x ≲2.4 compositions undergo a phase transition upon being heated to spacegroup Cmcm that involves disordering of the oxygensubstructure. Thermal expansion coefficients are reported for theseries. Complex impedance studies show that the Ba-rich samples aremixed proton and oxide ion conductors under moist atmospheres butare predominantly oxide ion conductors at high temperatures or underdry atmospheres. Sr-rich samples show significantly less water uptakeand appear to be predominantly oxide ion conductors under the conditionsstudied.We report the synthesis, structuralcharacterization, and oxideion and proton conductivities of the perovskite-related Ba Structural families of interest for oxide ion conductivityinclude fluorite derivatives,6 apatites,9 melilites,13 La2Mo2O9,15 and others. Perovskite derivatives such as ABO3−δ and brownmillerites A2B2O5 areof interest for both oxide ion and proton conductivity.Materials displayingeither proton or oxide ion mobility are crucialfor several important applications: as electrolytes in fuel cells,in sensors, and as ion-permeable membranes.3OhTd2O7.5, where A is a 2+ cation andOh and Td are octahedrally and tetrahedrally coordinated 3+ cations,respectively . The formal relationship to perovskiteis shown in V = 12Vperovskite created bythe transformation . Subsequentoxygen shifts and polyhedral rotations lead to the A3OhTd2O7.5 topology that has an interrupted network builtfrom corner-sharing octahedra and tetrahedra.17 The structure can be described as containing layers two polyhedrawide of fully connected ∞2[(OhO6/2)(TdO4/2)]4– alternating with layers containing Ga2O7 groups.In recentwork, we have shown high proton and oxide ion conductivityin a family of perovskite-related compounds with composition Aectively 1.16 Rela2O7 tetrahedra are not shared withother frameworkpolyhedra, so the Niggli description becomes A3(OhO6/2)(TdO4/2)(TdO3/2O). This frameworkbreaking is a natural consequence of the high Td content. A secondinteresting feature of the Td2O7 layers is thatthey show an obvious potential mechanism for oxygen positional disordervia a correlated motion of TdO3/2O groups. Similar motionshave been shown to enable high ion mobility in several materials.35 For example, one can imagine the pink Td–O–Td oxygenon the right of 2O7 groups by half of a unit cell vertically.Locally it could occur by coupled rotations of the underlinked TdO3/2O tetrahedra. We have previously shown16 that this type of transition occurs in alternate Ga2O7 layers at high temperatures in Ba3YGa2O7.5, leading to a change in Td2O7 ordering from the “Abakumov type”17 to the “Tamazyan type”.36 We have also shown that it has implicationson high-temperature ionic conductivity, with a significant increasein conductivity around the phase transition (TdO4/2) slabs. Coupled rotations of the octahedra and tetrahedrathen allow A cations to optimize their bonding and cause the Td2O7 groups to distort from the staggered configurationof In fact, oxygen disorder leadingto 50% occupancy of the pink andgray sites would give rise to disordered chains of TdOx3–SrxYGa2O7.5 with 0 ≤ x ≤ 3, where weobserve a family rich in structural transitions as a function of bothcomposition and temperature. The average structure across the seriesis studied using powder X-ray and neutron diffraction data, and thelocal structure and defects are probed using HAADF-STEM imaging. Impedancespectroscopy has been used to measure the oxide and proton conductivityacross the series, and the properties are correlated with structuralchanges.In thispaper, we explore the structural chemistry and physicalproperties of the new series of compounds Bax3–SrxYGa2O7.5 series  were synthesized from stoichiometricamounts of BaCO3 , SrCO3 , Y2O3 , and Ga2O3 .Starting compounds were ground together in ethanol, pelletized, andheated in alumina crucibles at 1200 °C for 12–18 h. Sampleswere reground, repelletized, and reheated until no changes in thepowder X-ray diffraction pattern occurred. Total heating times amountedto roughly 150 h at 1200 °C. All of the prepared phases formedwhite powders.Twogram polycrystalline samples of the Baa = 5.431195(9) Å at room temperature] was used foraccurate cell parameter determination. Analysis of the diffractiondata was performed using the Rietveld method37 implemented in the Topas Academic software.39 Structures were drawn using Crystalmaker or Vesta.40A Bruker D8 Advancediffractometerin Bragg–Brentano geometry with Cu Kα radiation and aLynx-eye PSD detector was used for all X-ray powder diffraction measurements.A 2θ range of 10–120° and collection times of ≤6h were used for final patterns, and an internal Si standard  witha β angle very close to90.0° [refined value of 88.997(4)°]. This has 47 structuralparameters and an Rwp of 4.4%. However,as discussed above, this P2/c modeldoes not fit the data perfectly. To improve the fit any further, thenumber of parameters needed to almost double to 91, and even the P1 model [with 177 structural parameters ] did not fully explain all of the diffractionfeatures observed.The best model identified bythis process was the monoclinic A centeringin the setting used ]. These P-only peaks are found to be significantlybroader than others, suggesting only short-range order. They werealso not visible in the laboratory X-ray data, suggesting a significantoxygen or lighter atom contribution. We could not identify any modelthat simultaneously described the P-only neutronpeaks without significant overcalculation of their X-ray intensity.We therefore also investigated structures with different Ga2O7 ordering patterns, including the Tamazyan-type orderingidentified for Ba3YGa2O7.5 at hightemperatures.16 None of these gave a significantimprovement in fit. Because average crystallographic methods failed,the structure was further investigated using HAADF-STEM imaging asdiscussed in the next section.Some insight into why none of the simpleaverage structural modelsfully explains the diffraction data can be gained by examining theweak peaks that violate cell centering [x = 2 (BaSr2YGa2O7.5),the structure remains atleast metrically orthorhombic, but diffraction data can no longerbe fitted by P2/c models. Inspectionof the X-ray diffraction pattern reveals that k + l = odd reflections are not present, and thus, the unitcell is centered. However, the Amma/Cmcm aristotype structure of C2 structureused by Wang et al. for Sr3YAl2O7.5.48 This gave a much better fit, withall observed peaks accounted for and β being refined to 90.015(4)°. The most appropriate spacegroup for BaSr2YGa2O7.5 might thereforebe a subgroup of Cmcm and a supergroup of C2. The possible orthorhombic space groups Ama2, Amm2, and C2221 andmonoclinic groups C2/c and C2/m were identified from the subgrouptree in Figure S4. BaSr2YGa2O7.5 was found to be SHG-active and thus has anoncentrosymmetric structure. C2/c, C2/m, and Cmcm can therefore be ruled out because they are centrosymmetric. Modelsin space groups Amm2 and C2221 fail to fit several reflections in the XRD pattern, whereas Ama2 and C2 give much better and essentiallyindistinguishable fits and plausible structural models. The Ama2 model requiresthe Ga2O7 groups to be disordered. While thiscould occur , the low-temperature orderedstructures of other compositions in the family suggest at least localorder is more likely. We therefore favor an ordered C2 monoclinic structure with a β very close to 90° at roomtemperature. The final C2 model was refined againsta 6 h XRD pattern with Ba and Sr distributed randomly across all Asites and is reported in Table S1.As the Sr content increases to x ≥ 2.4 Ba0.6Sr2.4YGa2O7.5, Ba0.15Sr2.85YGa2O7.5, and Sr3YGa2O7.5 compounds were all found to be isostructural, withWang’s Sr3YAl2O7.5 compoundin space group C2.48 Thismodel was refined against X-ray and neutron diffraction data of Sr3YGa2O7.5 to give the final fits shownin Figure S10 and the coordinates listedin Table S2. Views down different axesare shown in panels d and e of C2is supported by SHG measurements on Ba0.15Sr2.85YGa2O7.5, where a positive signal indicateda noncentrosymmetric structure.The x = 0.0, 1.5, 2.0, and 3.0 samplesto confirm the structural conclusions described above and gain moreinsight into local disorder, particularly for Ba1.5Sr1.5. The heavy atom columns in these structures emphasizedin panels b and e of a-axis of the Abakumov structure .We will use the Abakumov cell setting throughout this discussion;the Wang model would have space group A2 in thissetting. Because there is no unique way of matching labels betweenchemically similar sites of these two models, we use the Abakumovnumbering scheme and refer to Wang labels where needed.HAADF-STEM imagingwas performed on 2O7 groups around the Ga–O–Ga vector between adjacent(OhO6/2)(TdO4/2) two-dimensional slabs and theimpact this has on the relationship between polyhedral tilts in adjacentslabs. In the Sr3.0 structure of 2O7 groups in sequential layers from left to right arerelated by translational symmetry such that all of the cyan “terminal”oxygens of the closest tetrahedron are oriented to the top right.In contrast, for the primitive Ba3.0 structure  isbonded to two GaO3/2O terminal oxygens and four oxygensthat these Ga3/2O tetrahedra share with YO6/2 octahedra that must tilt cooperatively as the tetrahedra rotate.This means that the Ba2 position along the b-axisis anticorrelated with the terminal O position: when the terminaloxygen points “up” in y coordinatebecomes smaller and shifts “down” and vice versa. Muchsmaller shifts are observed for the other A site (Ba1) in the Ga2O7 layers. The pattern of A site shifts observedfor the end member structures is then as shown in panels a and d of 3.0 and down, down, down (↓↓↓) forSr3.0 along the long axis.This structural change impacts the nearbyA site coordination environments,especially those of A sites lying within the GaZ Aatoms (Ba or Sr), and the lighter ones correspond to either Ga/Gain columns of Ga2O7 groups or Y/Ga columns inthe (YO6/2)(GaO4/2) slabs. In each case, theimage quality has been enhanced by averaging over multiple equivalentparts of a larger-area image . The principal axes of Ga/Ga ellipses areconstrained by symmetry to lie along the c-axis.The lower portions of panels a and d of 6/2)(GaO4/2) slabs appear as essentially circular features centered on the horizontalyellow dashed lines. Finally, we see that the Ba1/Ba2 columns (Wang’sSr3/Sr4) appear elongated parallel to the b-axisand show characteristic shifts of ↓↑↓ relativeto the yellow dashed line for the Ba3 case and ↓↓↓for Sr3.0. The local structure is therefore in full agreementwith the powder crystallography.The Ba3/Ba4 columns inside the . Lineslabeled a and b, for example, show the opposite displacement to thatexpected on the basis of the line to their left. Interestingly, linea contains a significant number of orange columns where the displacementdirection could not be determined. This is presumably related to disorderwithin those atomic columns as they run into the page. The prevalenceof planar defects is highlighted in Z) A-site regions. We also saw evidence forthis type of short-range ordering in the broader P-only powder diffraction peaks discussed above.Panels b and e of Supporting Information).While these defects were common in Ba1.5Sr1.5, they were not observed in Ba1.0Sr2.0.Panels b and c of 2SrYGa2O7.5, which showedevidence for water loss on first heating .162.85Sr0.15YGa2O7.5 and Ba2.7Sr0.3YGa2O7.5 undergo a phase transition similarto that of Ba3YGa2O7.5  onheating. The phase transition temperature decreases smoothly withan increase in Sr content. Above the phase transition, the diffractionpatterns can be fitted well by the disordered Cmcm model of 2O7 ordering in Ba3YGa2O7.5 arenot as apparent in either of these compositions. There is, however,some diffuse scattering in similar positions in the high-temperatureXRD patterns of both compounds, hinting that some short-range Tamazyan-typeordering may be present . It seems likely that faults in planes perpendicular to the longunit cell axis broaden and weaken these peaks.BaYGa2O7.5 S11 onhex ≤ 1.5, there areno obvious changes in peak positions orintensities in the raw diffraction data; however, the cell parametertemperature variation suggests a phase transition in all three compounds.In particular, we see a reduction in thermal expansion along the b-axis at high temperatures, similar to that observed inBa3YGa2O7.5. The temperature at whichthe change occurs increases with Sr content, with Tc values of 370, 420, and 700 °C for x values of 0.6, 1.0 and 1.5, respectively. All of the diffractionpatterns across the temperature range were Rietveld-fitted with both P2/c and Cmcm models withatomic positions fixed at the values refined against the Ba3YGa2O7.5 diffraction data at 25 and 1000 °C,respectively; Rwp is plotted versus temperaturein Figure S15. The temperatures at whichthe Cmcm model becomes as good as P2/c correspond to the proposed phase transitiontemperatures, suggesting that the structure might undergo a transitionto the Cmcm model at high temperatures. However,we do not have neutron diffraction data at high temperatures for thesecompositions, so the nature of this transition cannot be stated definitively.For the metricallyorthorhombic 0.6 ≤ x = 2 BaSr2YGa2O7.5 compound also has a phase transition, occurring at ∼770°C on heating and cooling. This phase transition is characterizedby the disappearance of four weak peaks in the XRD pattern between2θ values of 25° and 45° . These are the same peaks that couldnot be indexed by a Cmcm model, suggesting the transitionis between the low-temperature C2 structure and thehigh-temperature disordered Cmcm structure.The x > 2 compositions, we observe smooth increasesin cell parameters as a function of temperature and no evidence ofmajor structural changes. Samples remain monoclinic (β ≈90.6°) up to at least 1000 °C.For x values of 0.0, 0.3,1.0, 1.5, 2.0, and 3.0 on heating and cooling from room temperatureto 1000 °C under dry air. Experiments were also performed on x = 0.0, 1.5, and 3.0 samples after deliberate exposureto moist atmospheres for an extended period. The results are shownin Figure S17 and supported by the protonNMR spectra in Figure S18. The least basic x = 3.0 Sr3YGa2O7.5 showsminimal water uptake upon exposure to atmospheric moisture, with smallmass losses consistent with surface adsorption. Ba-containing samplesshow more significant uptake, with the x = 0 compositionbecoming X-ray amorphous after exposure to moisture for 3 days andshowing a 19.5% mass loss on heating. More minor water uptake (∼0.5%)occurs during the ∼4 h period as samples are furnace-cooledfrom high temperatures. This is consistent with the “humps”observed around 500 °C in the unit cell plots in x values of 0 and 2. We note that no sucheffects are observed for Sr3YGa2O7.5.Thermogravimetricanalysis was performedon freshly heated samples with 2 to measure the conductivity of representativecompositions. Data for individual compositions are shown in Impedance spectroscopy was performed inair and dry Nx ≤ 0.3 P2/c compounds all show behavior similar to that of Ba3YGa2O7.5. There is a significant conductivityjump at the P2/c to Cmcm phase transition, which becomes more prominent with higher Sr content.The Ba-rich 2 at low temperatures, and the low activationenergy at low temperatures against 1/T are observed, reflecting changes in activationenergy. This is consistent with the smaller structural changes observedat the phase transition by PXRD. In particular, there is no abruptcell volume increase for these compositions .Each composition shows a proton contribution to conductivity atlow temperatures, evidenced by the difference in conductivity in airand dry Neratures 9c. At hiobserved 9c. This x ≥2 compositions exhibitslightly different behavior, in line with the change to a C2 structure. They have higher activation energies for ionicmigration at all temperatures than the x < 2 compositions.The small low-temperature proton contribution to conductivity decreaseswith an increase in Sr content, consistent with the diminishing tendencyto incorporate water. The conductivity of the x =2 composition, with the C2 structure (90°),is higher than the others, which all have comparable conductivitiesat 1000 °C. This difference cannot be accounted for by the pelletdensity difference (90% vs 88%) and is therefore likely to be structuralin origin. Compositions close to the boundary between two distinctstructures often have unusual microstructures, which can influencethe properties.The remaining tH, was calculated at 600 and 1000 °C for each composition usingwet and σdry are the measured conductivities in wet and dry atmospheres,respectively. This assumes dominant ionic conduction under both conditions.The results are plotted in 3YGa2O7.5 to incorporate water.At 1000 °C, the proton transport numbers are much lower, andoxide ion conductivity dominates. Ba3YGa2O7.5 is an exception with an approximate half-order-of-magnitudeincrease in conductivity in air across the whole measured temperaturerange. As discussed elsewhere,16 this suggestsa proton contribution remains at high temperatures.x3–SrxYGa2O7.5 systemexhibits a rich structural chemistryas a function of both composition and temperature that correlatesstrongly with ionic conductivity. We can rationalize many of the changeson the basis of the different bonding requirements of Ba and Sr. From 6/2)(GaO4/2) slabs relative to theuntilted Cmcm aristotype. This is analogous to theoctahedral tilting seen in ABO3 perovskites to optimizebonding of the ≤12-coordinate A site. In the A3OhTd2O7.5 structure, the maximum A site coordinationnumbers differ due to the oxygen deficiency relative to perovskiteand are summarized for the four unique A sites in e, 2f, 4g, and 4g Wyckoff positions),we find the average A site coordination number is 10, as expectedfor an ABO2.5 composition relative to 12-coordinate A inan ABO3 perovskite. Because assigning formal coordinationnumbers and average bond lengths for Ba and Sr is somewhat arbitrary, The Ba6/2)(GaO4/2) slabs have highercoordination numbers and correspondingly longeraverage bond lengths compared to those of Ba1 and Ba2. We also seethat the polyhedral tilting upon introduction of the smaller Sr leadsto more distorted A site coordination environments. This is shownby the variance of the important Sr–O bond distances beingapproximately 3 times that of Ba–O for each site. It is interestingto note that for all sites except the strictly eight-coordinate A2,several A–O distances increase upon introduction of the smallerSr2+. This is the corollary of the bond valence distortiontheorem discussed by Brown,49 i.e., thatcoordination environments distort to produce shorter and longer bondlengths to increase bond valence sums to expected values when a cationis too small for a given cavity. All of these observations are consistentwith the general trend for Sr2+ to exhibit coordinationnumbers lower than those of Ba2+ in oxides. Waroquier’sanalysis, for example, shows ∼21% of Sr2+ coordinationenvironments can be described as eight-coordinate compared to only9% for Ba2+.50We see from x = 0 to 0.6,the tilt pattern and P2/c structureof Ba3YGa2O7.5 are initially retained,though with a decreasing β angle as the Sr content increases.As the Sr content increases further, partial segregation of Sr tothe Ga2O7 layers will create regions of Sr-likestacking in the Ba structure, with the microdomain structure leadingto a metrically orthorhombic unit cell. At the Ba1.5Sr1.5 composition, these Sr-like layers are clearly visible inthe HAADF-STEM images, even though the structure remains primitive.By Ba1.0Sr2.0, the smaller average A size favorsthe centered structure, and the lack of defects observed by HAADF-STEMis consistent with the Ga2O7 layers having aconsistent Sr-rich occupancy. The increasing relative stability ofthe centered structure with x leads to an initialdecrease in the phase transition temperature upon heating of low-x samples plotted in x = 3. A similar decrease in the phase transitiontemperature was reported by Abakumov et al. as the size of Oh cationswas increased in Ba3OhTd2O7.5  compounds.17The lowereffective coordination numbers of site Ba1 and Ba2 then imply a tendency for Sr segregation on these positions, consistentwith the HAADF-STEM observations. This helps explain the structuralevolution in 2– migration involving reorientationof Ga2O7 tetrahedra. High-temperature diffractionexperiments show phase transitions associated with either reorientationof these tetrahedra (at low x) or their local disorder,and conductivity data show changes at the corresponding temperatures.For x = 0, 0.15, and 0.3, we see abrupt changes inconductivity at Tc related to abrupt volumechanges. For x > 0.3, the unit cell volume changescontinuously at Tc, and the conductivitychanges more gradually. The importance of the structure of the Ga2O7 layer to conductivity is further highlightedby the increase in the ion migration activation energy at x = 2, where the structure changes from primitive to centered,which is associated with local changes in the Ga2O7 environment. The larger cell volume of Ba-rich samples alsoallows significant water uptake leading to a large proton conductivitycontribution that is largest around 350–500 °C, beforeH2O is lost at higher temperatures.The observed evolution of conductivity across the series canberelated to these structural changes. The arrangement of octahedra,tetrahedra, and vacant oxygen sites in the structure see 4 suggestx3–SrxYGa2O7.5 solid solution range and exhibita complex structural phase diagram as a function of composition andtemperature. Compounds at the Ba- and Sr-rich compositional extremeshave P2/c and C2 structures, respectively, at room temperature. As the Sr contentis increased from x ≈ 0 to compositions suchas Ba2.4Sr0.6YGa2O7.5 andBa1.5Sr1.5YGa2O7.5, HAADF-STEMimaging reveals intergrowths of Sr-like regions in the Ba structuretype. The resultant microstructure leads to a metrically orthorhombicunit cell. For x = 2 Ba1Sr2YGa2O7.5, diffraction data, STEM imaging, andSHG measurements suggest a structure in space group C2 with a ∼90° monoclinic angle. Most of the compoundsin the series appear to undergo a transition to a higher-symmetry,disordered Cmcm-like structure at high temperatures,except for Ba3YGa2O7.5, which adoptsan alternative Ga2O7 ordering pattern, and the x > 2 Sr-rich compositions where no phase transitionisobserved up to 1000 °C.In conclusion, single-phase materials can be prepared across thewhole Bax =0 to x = 1.5. From x = 2, thereis an abrupt increase in the activation energy for ion migration,corresponding to the structural change to C2. Thecontribution of protons to conductivity peaks in the middle of theseries before decreasing as the Sr content increases. Unlike the casefor Ba3YGa2O7.5, TGA and proton NMRexperiments showed Sr3YGa2O7.5 takesup very little water and is stable in moist atmospheres. The Sr-richcompounds are therefore of particular interest for further developmentas solid electrolyte materials.Impedance spectroscopy shows thatall members of the family showeither oxide ion or proton conductivity, depending on the experimentalconditions. There is a general decrease in conductivity as the Srcontent increases, consistent with the smaller unit cell volumes.Conductivity properties evolve smoothly from


Caesalpinia bonduc (Linn.) Roxb. In this study, we investigated the effect of caesalmin C on Aβ-induced toxicity and possible mechanisms in the transgenic Caenorhabditis elegans AD model. Our results showed that caesalmin C significantly alleviated the Aβ-induced paralysis phenotype in transgenic CL4176 strain C. elegans. Caesalmin C dramatically reduced the content of Aβ monomers, oligomers, and deposited spots in AD C. elegans. In addition, mRNA levels of sod-3, gst-4, and rpt-3 were up-regulated, and mRNA levels of ace-1 were down-regulated in nematodes treated with caesalmin C. The results of the RNAi assay showed that the inhibitory effect of caesalmin C on the nematode paralysis phenotype required the DAF-16 signaling pathway, but not SKN-1 and HSF-1. Further evidence suggested that caesalmin C may also have the effect of inhibiting acetylcholinesterase (AchE) and upregulating proteasome activity. These findings suggest that caesalmin C delays the progression of AD in C. elegans via the DAF-16 signaling pathway and that it could be developed into a promising medication to treat AD.Alzheimer’s disease (AD) is the most prevalent neurodegenerative disease in the world. However, there is no effective drug to cure it. Caesalmin C is a cassane-type diterpenoid abundant in  Alzheimer’s disease (AD) is a progressive, irreversible neurodegenerative disease. It is clinically characterized by cognitive decline, memory loss, and executive function impairment. AD accounts for about 55–75% of dementia, and the incidence of AD increased dramatically along with the aging of society’s population . It is e25–35 aggregation through autophagy pathway and through inhibition of oxidative stress ; CL2006, dvIs2 [pCL12 (unc-54/human Aβ peptide1−42minigene) + pRF4]; CL2355, dvIs50 [pCL45 (snb-1::Abeta 1–42::3′ UTR(long) + mtl-2::GFP]; CL2122, dvIs15 [(pPD30.38) unc-54(vector) + (pCL26) mtl-2::GFP]; CL2179, dvIs179 [myo-3p::GFP::3′ UTR(long) + rol-6(su1006)]; TJ356, zIs356 [daf-16p::daf-16a/b::GFP + rol-6]; CL2166, dvIs19 [(pAF15) gst-4p::GFP::NLS] III; CF1553, muIs84 [(pAD76) sod-3p::GFP + rol-6(su1006)]; GR2183, mgIs72 [rpt-3p::GFP + dpy-5(+)] II. All C. elegans strains and Escherichia coli OP50 were purchased from the Caenorhabditis Genetics Center . Worms of CL4176, CL2355, CL2122, and CL2179 were cultured on nematode growth medium (NGM) plates seeded with E. coli OP50 as food resources in an incubator at 15 °C; the other strains were grown at 20 °C. To harvest age-synchronized animals, pregnant adults were treated with hypochlorite and eggs were washed three times with M9 buffer.1–42 gene containing a 3′UTR sequence under the body wall muscle myo-3 promoter. This strain contains the temperature-sensitive gene, smg-1. At permissible temperatures, the transcription product of the 3′UTR was degraded and the Aβ peptide was barely expressed. When the temperature rose to 25 °C, the smg-1 gene was functionally inactivated, the Aβ transcript was stably expressed, and a large amount of Aβ protein accumulated in the nematode body wall muscle, resulting in nematodes showing paralytic symptoms [CL4176 transgenic nematodes regulate Aβ peptide expression by transferring an Aβsymptoms .Synchronized eggs were incubated at 15 °C for 30 h and then transferred to fresh NGM plates containing caesalmin C 100 µM, 200 µM, and 400 µM, respectively. A mixture of 0.1% DMSO and 0.3% PEG400 was used as solvent control. When the nematodes reached L3-stage, the incubation temperature was changed from 15 °C to 25 °C. After 28 h, paralyzed individuals were counted every 2 h. When lightly touched with a platinum wire ring, the nematodes were immobile. If only the head could move, it was considered paralyzed. Three independent biological replicates were performed in this experiment.1–42 gene was transferred under the snb-1 promoter in neurons. After treatment at 25 °C, smg-1 was functionally inactivated and Aβ protein was expressed in large numbers in nematode neurons, resulting in a substantial reduction in tolerance to exogenous 5-HT in this strain of nematodes, which eventually exhibited a state of rigidity [CL2355 transgenic nematodes also contained the smg-1 temperature monitoring system, and the human-derived Aβrigidity .Synchronized L1 larvae were transferred to fresh NGM plates, with or without caesalmin C amounts of 100 µM, 200 µM, and 400 µM, treated at 15 °C for 65 h, then transferred into a 25 °C incubator for 30 h. The nematodes were collected in M9 buffer, and the number of paralytic nematodes was counted on 96-well plates treated with 5 mg/mL of serotonin for 5 min. CL2122 was used as a transgenic control, and this experiment was repeated at least three times.As a control strain of CL4176 nematodes, CL2179 nematodes were transferred only to green fluorescent protein (GFP) under myo-3 promoter. This strain of nematodes expresses GFP in large amounts in muscle cells when induced at 25 °C, resulting in a strong green fluorescence around the body. Synchronized eggs of CL2179 were incubated at 15 °C for 30 h, and then seeded on fresh NGM plates with or without caesalmin C in amounts of 100 µM, 200 µM, and 400 µM. They were treated at 15 °C for 67 h, followed by 25 °C for 30 h. The nematodes were washed with M9 buffer and collected. The nematodes were fixed in the center of the slide with 20 mM sodium azide, covered with a coverslip, and the overall fluorescence intensity of the nematodes was observed and recorded under a fluorescence microscope . At least 30 nematodes were recorded in each treatment group. The fluorescence intensity of the whole body of the nematode was quantified with ImageJ software.1–42 protein slowly in muscle cells when cultured at 20 °C, resulting in progressive nematode paralysis. Synchronized eggs were incubated at 20 °C for 30 h, and then transferred to fresh NGM plates with or without caesalmin C amounts of 100 µM, 200 µM and 400 µM, followed by treatment at 20 °C for 6 days. The nematodes were then collected in M9 buffer and washed three times, after which 4% tissue cell fixative was added and placed in a 4 °C refrigerator for 24 h. The supernatant was removed by centrifugation and placed at 37 °C for 24 h after the addition of 125 mM Tris–HCl (pH 7.4) with 1% TritonX-100 and 5% β-mercaptoethanol. Aβ deposits were dyed with 0.125% thioflavin S (ThS) in 50% ethyl alcohol and decolored background with 50% ethyl alcohol. Fluorescence images were taken from the same parameters under a fluorescence microscope . The fluorescent spots in front of the pharyngeal bulb of each individual were counted using ImageJ software. Each group contained at least 30 individuals, and the experiment was performed in three independent biological replicates.Transgenic CL2006 strain nematodes expressed human-derived Aβ® III Reverse Transcriptase Kit , respectively. Real-time PCR was performed using the Hieff® qPCR SYBR Green Master Mix  and applied real-time fluorescence quantification instrument . The relative quantities of mRNA were determined using 2–ΔΔCt methods and normalized against the housekeeping gene (β-actin) mRNA. The sequences of forward and reverse primers used were: CCGACATGACTCAGGATATGAAGT and CACCATGAGTCCAATGATTGCA for Aβ; AGTGGGCTCCTGTTCGAGAA and CCAATAGAAAATCACCATCGACAA for ace-1; CAATAATCAACTCATGGGCATCA and TTTTCGCGAGACGAAACGA for ace-2; TCCCCATACGAAACAACACA and CTCCTCCCAGCTTTTCCACAA for TNFA1P; CCAGAAGAATCCCCATACGA and TCCTCCTCCAACTTTTCCAAA for TNFA1P1; CGTAGGCGATCTAGGAAATGTG and AACAACCATAGATCGGCCAACG for sod-1; AGCTTTCGGCATCAACTGTC and AAGTCCAGTTGTTGCCTCAAGT for sod-2; TTCAAAGGAGCTGATGGACACT and AAGTGGGACCATTCCTTCCAA for sod-3; GTTGTCTAAGTGCTGGTGG and TTCCACATGCAAGTCGGCT for sod-4; GCTGAAGCCAACGACTCCAT and GACCGAATTGTTCTCCATCGA for gst-4; CCACGTCATCAAGGAGTCAT and GGAAGCGTAGAGGGAGAGGA for β-actin.Synchronized L1 larvae of CL4176 were transferred to fresh NGM plates with or without caesalmin C and incubated at 15 °C for 65 h, followed by induction at 25 °C for 30 h. Nematodes were collected with M9 buffer and washed three times. RNA extraction and cDNA synthesis were performed according to the manufacturer’s protocols of TRIeasyTM Total RNA Extraction Kit  and HifairE. coli HT115 cloned target gene of hsf-1, daf-16, and skn-1, or only empty L4440 vector. Synchronized eggs were transferred to NGM plates with or without the caesalmin C and treated at 15 °C for 48 h, followed by induction at 25 °C for 30 h. Paralytic nematodes were scored according to the method of the paralysis experiment.Transgenic CL4176 nematodes were cultured on fresh NGM plates seeded with interfering bacteria for two generations and then collected for synchronization. Respectively, the RNAi NGM plates contained 100 μg/mL ampicillin, 5 μg/mL tetracycline hydrochloride, and 1 mM isopropyl β-D-thiogalactopyranoside, and were seeded with The TJ356 strain integrates DAF-16 with GFP by gamma irradiation. Normally, this strain of nematodes shows a uniform green fluorescence around the body, and if the compound activates DAF-16, the DAF-16 nuclear transcription factor translocated from the cytoplasm to the nucleus, resulting in bright aggregation sites in nematodes.Synchronized L1 larvae of TJ356 were transferred to fresh NGM plates with or without caesalmin C and treated at 20 °C for 62 h. Afterward, nematodes were collected in M9 buffer and washed three times. The nematodes were fixed in the center of the slide with 20 mM sodium azide anesthesia, and the percentage of nematodes with nuclear translocation in each group was observed and counted under a fluorescence microscope . At least 30 nematodes were recorded in each treatment group, and the experiment was repeated three times independently.v/v), 10% glycerol (v/v), 4% β-mercaptoethanol (v/v), and 1× protease inhibitor) at 100 °C for 10 min, followed by ice bath cooling for 20 min and centrifugation at 4 °C for 10 min at 14,000× g. The supernatant was then aspirated and added to an appropriate amount of 5× loading buffer, mixed well and boiled for 10 min at 100 °C in a metal bath, then stored at −80 °C. The membrane was blocked at room temperature with 5% milk in TBS-Tween and then incubated with primary antibody  overnight at 4 °C. β-actin  was used as an internal reference control and goat anti-mouse HRP  was used as a secondary antibody. Finally, images were taken from the Tanon ECL detection system  and quantified by ImageJ software. The molecular weights of the Aβ monomer and oligomer were 4 kDa and 20 kDa, respectively. The relative densitometry analysis normalized to β-actin values.Protein extraction was performed according to the previously disclosed method . Nematod2 catalyzed by xanthine oxidase produced superoxide anion, which reduces NBT to formazan with strong absorption at 560 nm. However, SOD scavenges the superoxide anion and thus inhibits the formation of formazan, so the higher the SOD activity, the lower the absorbance value. Accordingly, the effect of the compound on SOD activity can be analyzed by comparing the absorbance values at the same tissue concentration.Nitro-blue tetrazolium (NBT) is an alkaline phosphatase substrate. The reaction between xanthine and OSynchronized L1 larvae of CL4176 were received on fresh NGM plates with or without caesalmin C, treated at 15 °C for 67 h, and then induced at 25 °C for 30 h. The nematodes were collected with M9 buffer and snap-frozen in liquid nitrogen, and the homogenate was made by adding the appropriate amount of pre-cooled PBS solution at 4 °C for ultrasonic crushing, followed by centrifugation at 4 °C for 10 min, and the supernatant was taken as a reserve. The protein content in the supernatant of each group was determined using the BCA method. The SOD activity in the supernatant of each group was measured according to the instructions of the NBT kit . Relative absorbance values were normalized by protein concentration. Referring to the method described in the literature , the eff.rpt-3::GFP fusion protein, and the intensity of green fluorescence was enhanced in nematodes when the compound was able to promote the expression of the proteasome subunit rpt-3. Synchronized L1 larvae of GR2183 were received on fresh NGM plates with or without caesalmin C, and treated at 20 °C for 3 days. Each group of nematodes was collected and washed three times with M9 buffer. These nematodes were fixed in the center of the slide with 20 mM sodium azide, observed, and recorded under a fluorescent microscope . The fluorescence intensity of each group of nematodes was analyzed by ImageJ software, and the results were expressed as histograms. At least 30 nematodes in each group were subjected to three independent experimental replicates.Transgenic GR2183 nematodes were transfected with p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05, “ns” means no significant difference). The Kruskal-Wallis test was used if the data did not conform to normal distribution. Between-group differences in paralysis data were statistically analyzed using the Kaplan-Meier and log-rank tests for survival analysis in SPSS software. All results were plotted using GraphPad Prism 9.0.Statistical analysis was performed using SPSS 23.0 software. Statistical differences were determined using the one-way ANOVA, with the mean ± SD indicating data, and asterisks were used to indicate significant differences (**** gst-4 and antioxidant gene sod-3, thereby strengthening stress resistance. The pro and con results showed that caesalmin C resistance to Aβ-induced toxicity required the involvement of the DAF-16 signaling pathway, but not the SKN-1 and HSF-1 signaling pathways. In addition, caesalmin C may also resist Aβ-induced toxicity by inhibiting AchE activity and activating the ubiquitin-proteasome pathway. The potential signal mechanisms involved are shown in In summary, caesalmin C activated partial nuclear translocation of DAF-16 and up-regulated the expressions of detoxification gene


N6-etheno-ATP (eATP) at the anti-luminal side of nondistended (empty) or distended (full) murine (C57BL/6J) detrusor-free bladder model, using HPLC. The hydrolysis of eATP and eADP was greater in contact with LP of distended than of nondistended bladders whereas the hydrolysis of eAMP remained unchanged during filling, suggesting that some steps of eATP hydrolysis in the LP are mechanosensitive. eATP and eADP were also catabolized in extraluminal solutions (ELS) that were in contact with the LP of detrusor-free bladders, but removed from the organ chambers prior to addition of substrate. The degradation of both purines was greater in ELS from distended than from nondistended preparations, suggesting the presence of mechanosensitive release of soluble nucleotidases in the LP. The released enzyme activities were affected differently by Ca2+ and Mg2+. The common nucleotidase inhibitors ARL67156, POM-1, PSB06126, and ENPP1 Inhibitor C, but not the alkaline phosphatase inhibitor (-)-p-bromotetramisole oxalate, inhibited the enzymes released during bladder distention. Membrane-bound nucleotidases were identified in tissue homogenates and in concentrated ELS from distended preparations by Wes immunodetection. The relative distribution of nucleotidases was ENTPD1 >> ENPP1 > ENTPD2 = ENTPD3 > ENPP3 = NT5E >> ENTPD8 = TNAP in urothelium and ENTPD1 >> ENTPD3 >> ENPP3 > ENPP1 = ENTPD2 = NT5E >> ENTPD8 = TNAP in concentrated ELS, suggesting that regulated ectodomain shedding of membrane-bound nucleotidases possibly occurs in the LP during bladder filling. Mechanosensitive degradation of ATP and ADP by membrane-bound and soluble nucleotidases in the LP diminishes the availability of excitatory purines in the LP at the end of bladder filling. This might be a safeguard mechanism to prevent over-excitability of the bladder. Proper proportions of excitatory and inhibitory purines in the bladder wall are determined by distention-associated purine release and purine metabolism.Prior studies suggest that urothelium-released adenosine 5′-triphosphate (ATP) has a prominent role in bladder mechanotransduction. Urothelial ATP regulates the micturition cycle through activation of purinergic receptors that are expressed in many cell types in the lamina propria (LP), including afferent neurons, and might also be important for direct mechanosensitive signaling between urothelium and detrusor. The excitatory action of ATP is terminated by enzymatic hydrolysis, which subsequently produces bioactive metabolites. We examined possible mechanosensitive mechanisms of ATP hydrolysis in the LP by determining the degradation of 1, While neural control of the bladder has been extensively investigated, local mechanisms of mechanotransduction from mucosa (urothelium) to nerves or detrusor during bladder filling have remained elusive  and voiding (micturition) of urine. Normal operation of these functions depends on proper detrusor excitability regulated by systemic  how distention of the bladder wall during bladder filling affects the hydrolysis of ATP and other purines in the LP and 2) the possible involvement of membrane-bound and soluble enzymes in these processes.2, 23.8 NaHCO3, 1.2 KH2PO4, 11.0 dextrose, and 1.8 CaCl2 (pH 7.4) and subject to further dissection.Adult (11–16 weeks) C57BL/6J male and female mice  were used in this study. Mice were euthanized by sedation with isoflurane followed by cervical dislocation and exsanguination. The urinary bladder was removed and placed in oxygenated ice-cold Krebs-bicarbonate solution (KBS) with the following composition (mM): 118.5 NaCl, 4.2 KCl, 1.2 MgClAnimals were maintained and experiments were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the Institutional Animal Use and Care Committee at the University of Nevada.Ex vivo bladder preparations with detrusor smooth muscle removed were prepared as described previously (eviously . BrieflyN6-etheno-derivatives of ATP (eATP), ADP (eADP), and AMP (eAMP) (2 µM each) were used as substrates. Reactions were performed at 37°C either in the presence of bladder preparations or in solutions collected from baths that previously contained bladder preparations . Following addition of substrate (0 min), 20 µl samples were collected from the reaction solution at 10 s, 2, 4, 6, 8, 10, 20, 30, 40, and 60 min and diluted 10-fold in ice-cold citric phosphate buffer (pH 4.0) to stop the enzymatic reactions. Collected samples were compared with a 2 µM substrate in KBS that has not been in contact with enzyme (designated as “beaker” sample). All samples were stored at −20°C until final analysis. Substrate decrease and product increase was evaluated by ultrasensitive HPLC-FLD methodology as described in N6-etheno-nucleotides. Each e-purine was expressed as percent of total purines detected in each sample. Only one substrate was used in each bladder preparation.1,2/5% CO2. After equilibration for 20 min, KBS in the chamber was replaced with fresh KBS and the bladder was either left empty (nondistended condition) for the time equivalent to filling or filled at 15 μl/min with KBS via an infusion pump  to ∼85–90% of bladder capacity determined at time of dissection (distended condition). Such filling volumes were similar to the volumes that were necessary to generate pre-voiding intravesical pressure  that was in contact with the bladder preparation was transferred to an empty water-jacketed chamber with an oxygen line. Substrate eATP, eADP or eAMP was added to the transferred solution at a final concentration of 2 µM and its degradation in the absence of tissue was evaluated for 1 h . Samples were concentrated by centrifugation at 4,000 × g for 25 min at 4°C using swing bucket rotor . The concentrated EL solutions (cELS) were brought to 200 µl with oxygenated KBS and enzymatic reactions were performed at 37°C by adding substrate eATP, eADP or eAMP to the cEL sample (2+ and/or Mg2+ in the absence or presence of 5 mM EGTA and/or 5 mM EDTA (2+-free solution (mKBS-A), 2) Ca2+-free KBS with 5 mM EGTA (mKBS-B), 3) Ca2+-free and Mg2+-free KBS with 5 mM EGTA (mKBS-C), 4) Ca2+-free KBS with 5 mM EGTA and 5 mM EDTA (mKBS-D), and 5) Ca2+-free and Mg2+-free KBS with EGTA and EDTA (5 mM each) (mKBS-E) (2+ (mKBS-A) or Ca2+ (mKBS-B and mKBS-E) and was compared with the eADP hydrolysis in regular KBS.Denuded bladder preparations were placed in water jacketed chambers filled with oxygenated KBS at 37°C. Following the equilibration, the KBS in the chamber was replaced with fresh KBS and the bladder was either left nondistended for the time equivalent to filling or it was filled (distended) at 15 μl/min with KBS to pre-voiding filling volume. This step ensured that soluble enzymes, if available, were released in regular KBS. Then, 2.9 ml of EL solution from nondistended or distended preparation was concentrated to approximately 80 µl using Amicon Ultra centrifugal filter units  as described in  mM EDTA  was adde(mKBS-E) . Time-coTo assess the effects of nucleotidase inhibitors, enzymatic reactions were conducted as described in Ex vivo bladder preparations with intact detrusor smooth muscle were placed in a dissecting dish with oxygenated KBS and pinned by the serosa to the bottom of the dish. A PE-20 catheter was placed through the urethra into the bladder lumen as described in Ex vivo Detrusor-Free Bladder Preparation. A linear probe for microdialysis (MD) with 2-mm dialysis membrane  was carefully implanted between the bladder mucosa and the detrusor , 1,N6-etheno-ADP (eADP), 1,N6-etheno-AMP (eAMP), and 1,N6-etheno-adenosine (eADO) were prepared according to a modified method of 2HPO4, was added to 150 µl of 200 µM authentic purine  in an Eppendorf tube. 2-Chloroacetaldehyde was synthesized according to a modified method of 2SO4 in distilled H2O and chloroacetaldehyde dimethyl acetal were added to a round bottom boiling flask. The mixture was distilled slowly under a fume hood and the distillate fraction containing approximately 1.0 M 2-chloroacetaldehyde was collected at 79–82°C. The reagent was stored at −20°C when not in use. Twenty µl 2-chloroacetaldehyde was added to the Eppendorf tube containing the mixture of authentic purine and citric buffer in a fume hood and then heated for 40 min at 80°C in a dry bath incubator  to produce 1,N6-etheno-purine substrates and 1,N6-etheno-purine standards  column and a matching direct-connect guard column . Gradient elution with 0.1 M KH2PO4 (pH 6.0) and increasing methanol (0–35% over 18 min) was employed. Column and autosampler temperatures were maintained at 25 and 4°C, respectively. Flow rate was 1 ml/min, run time was 20 min and post-run time was 5 min 1,N6-etheno-derivatized purines were detected by fluorescence at an excitation wavelength of 230 nm and emission wavelength of 420 nm  were processed with every set of samples.A reverse phased gradient Agilent 1,200 liquid chromatography system equipped with a fluorescence detector (FLD)  was used to detect 1,eviously . Brieflyf 420 nm . ChemSta™ GloMax®-Multi Detection System . LDH activity in samples was compared with a NADH standard and the assay was validated using a positive LDH control provided by the manufacturer. LDH activity was calculated according to the manufacturer instructions, reported in mU/ml and plotted as mean ± SEM.Lactate dehydrogenase (LDH) activity . The samples were mixed with the 5X Fluorescent Master Mix  and heated at 95°C for 5 min. The boiled samples, protein ladder, blocking buffer, primary antibodies, ProteinSimple horseradish peroxidase-conjugated anti-rabbit or anti-sheep (Invitrogen) secondary antibodies, luminol-peroxide, and wash buffer were loaded into the Wes plate . The plates and capillary cartridges were loaded into the Wes instrument, and protein separation, antibody incubation, and imaging were performed using default parameters. Compass software (ProteinSimple) was used to acquire the data, and to generate the virtual blot image reconstruction and chemiluminescence signal intensity electropherograms. The electropherogram shows the intensity detected along the length of the capillaries, and shows automatically detected peaks, that can be quantified by calculation of the area under the curve (AUC). The lane view is a virtual blot generated by the software from the actual data output, which takes the form of chemiluminescence signals versus apparent MW. Protein levels are expressed as the AUC of the peak chemiluminescence intensity.For automated capillary electrophoresis and Western blotting by Wes , mouse brain, urothelium tissue, and cELS were snap-frozen in liquid Nrocessed . cELS sarocessed . The homThe following primary antibodies were used for Wes analysis: from Cell Signaling Technology, rabbit anti-ENTPD1, and (E2X6B); rabbit anti-NT5E, (D7F9A); from ThermoFisher, sheep anti-ENTPD2, and (PA5-47777); rabbit anti-ENTPD3, (PA5-87888); rabbit anti-ENPP1, (PA5-17097); rabbit anti-ENPP3, (PA5-67955); from EpiGentek, rabbit anti-ENTPD8, and (A62482); from Abcam, rabbit anti-NT5C1A, and (ab190214); and, from Novus Biologicals, rabbit anti-TNAP, (SA40-00).Adenosine, ATP, ADP, AMP, chloroacetaldehyde dimethyl acetal, dimethyl sulfoxide (DMSO), and Triton X-100 , Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA) ; N,N′-1,2-Ethanediylbis [N-(carboxymethyl)]glycine (EDTA) , 6-[(3-aminophenyl)methyl]-N,N,5-trimethyl-triazolo pyrimidin-7-amine (ENPP1 Inhibitor C) , (-)-p-bromotetramisole oxalate (L-p-BT) ; 6-N,N-Diethyl-D-β,γ-dibromomethyleneATP trisodium salt (ARL67156), sodium metatungstate (POM-1), and 1-Amino-4-(1-naphthyl)aminoanthraquinone-2-sulfonic acid sodium salt (PSB06126) Data presented are means ± SEM. In some linear XY graphs, the data points are so similar that the symbols from individual points are superimposed. In some cases, the SEMs lie within the symbol. Scattered plot analysis was performed when appropriate. Means are compared by one-way ANOVA (Wes data) or two-way ANOVA (substrate degradation) for comparison of more than two groups followed by Dunnett’s, Tukey’s or Sidak’s multiple comparisons tests per GraphPadPrism, v. 8.4.2., GraphPad Software, Inc., San Diego, CA. A probability value less than 0.05 was considered statistically significant.n = 4 and P = 0.9638). The LDH activity was 7.04 ± 0.47% and 30.97 ± 10.95% in cELS of detrusor-free and intact preparations (p > 0.05) when compared with the LDH activity in Triton-X treated samples taken as 100%. This data suggests that the detrusor-free bladder preparation does not display greater cell damage than effects that normally occur in commonly-used ex vivo bladder dissection.LDH is a cytoplasmic enzyme present in most cells that is released upon damage of the cytoplasmatic membrane. We tested the LDH activity in cELS collected from detrusor-free preparations and from preparations with intact bladder walls as commonly used in bladder research. We compared the cell damage (measured as LDH activity) of the two types of preparations with cELS collected from detrusor-free bladders that were treated with Triton-X to induce a significant cell damage. The LDH activity was 186.39 ± 41.88 mU/ml in samples from Triton-X-treated preparations and 12.92 ± 2.69 and 50.53 ± 10.9 mU/ml in samples from distended detrusor-free and intact preparations, respectively (n = 5 in each group). In nondistended preparations, eATP comprised 94.19 ± 1.13% of total purines in beaker (0 min) and reached 45.39 ± 3.33% at 60 min (p < 0.0001) of reaction. In distended preparations, a significant decrease of eATP was observed at earlier time points than in nondistended preparations: eATP was reduced from 94.39 ± 0.9% of total purines in beaker to 14.55 ± 2.87% at 60 min (p < 0.0001) of reaction, 2wayANOVA with Dunnett’s multiple comparisons test. The eATP decrease in nondistended preparations reached statistical significance at 8 min, whereas in distended preparations the decrease of eATP was statistically significant at 2 min of reaction (p < 0.05). The eATP decrease was significantly greater in distended than in nondistended preparations for the entire time of reaction starting at 4 min (P = 0.0170), 2way ANOVA Sidak’s multiple comparisons test. The increase of the eATP products eADP, eAMP, and eADO was also greater in distended than in nondistended preparations. Thus, at 60 min of reaction, eADP was 23.54 ± 2.29% and 31.23 ± 2.03% (P = 0.0637); eAMP was 13.56 ± 0.98% and 25.31 ± 1.27% (p < 0.0001), and eADO was 17.5 ± 1.28% and 28.9 ± 4.09% (p < 0.0001) in nondistended and distended preparations, respectively; 2way ANOVA Sidak’s multiple comparisons test.To determine the degradation of eATP, eADP, and eAMP in contact with the LP, each substrate was added to the bath containing nondistended or distended detrusor-free bladder preparation. As shown in p < 0.0001) of reaction in nondistended preparations and from 87.56 ± 1.14% in beaker to 25.86 ± 4.4% at 60 min (p < 0.0001) of reaction in distended preparations, 2way ANOVA, Tukey multiple comparisons test. The eADP decrease was statistically significant at 10 min and at 30 min of reaction in distended and non-distended preparations, respectively (p < 0.05 vs. 0 min). The eADP decrease was greater in distended than in nondistended preparations during the enzymatic reaction starting at 6 min (P = 0.0153), n = 4 in each group, 2way ANOVA with Sidak’s multiple comparisons test. The increase of the eADP products eAMP and eADO was also greater in distended than in nondistended preparations. Thus, at 60 min of reaction, eAMP was 22.18 ± 1.29% and 33.79 ± 2.05% in nondistended and distended preparations, respectively (p < 0.0001) whereas eADO was 30.12 ± 5.19% and 40.35 ± 5.93% in nondistended and distended bladders, respectively (P = 0.0277).Similar results were obtained with eADP as substrate . eADP wan = 4 in each group, p < 0.0001 vs. 0 min, 2way ANOVA Tukey’s multiple comparisons test. No significant differences were observed in eAMP decrease or eADO increase between nondistended and distended preparations at all time points of reaction (p > 0.05), 2way ANOVA Sidak’s multiple comparisons test.The eAMP substrate  was decr). eATP was decreased from 91.56 ± 0.18% of total purines in beaker to 76.83 ± 4.84% (P = 0.0361) in dialysate from nondistended preparations (n = 8). eADP was 8.085 ± 0.18% and 12.87 ± 1.92% in beaker and dialysate, respectively (P = 0.0839) whereas eAMP was increased from 0.36 ± 0.002% in beaker to 6.88 ± 2.34% in dialysate (P = 0.0478). eADO was also significantly increased from 0% in beaker to 4.057 ± 0.596% of total purines in dialysate from nondistended preparations (p < 0.0005); one-way ANOVA with Dunnett’s multiple comparisons test. We also measured the degradation of eATP during microdialysis of a bladder that was filled with KBS at 15 μl/min. Similar to nondistended preparations, eATP degradation was observed in dialysates from distended preparations: thus, eATP was reduced to 77.58 ± 4.15% (P = 0.0237) and eADP, eAMP, and eADO were increased to 13.13 ± 1.65% (P = 0.0403), 6.66 ± 1.94% (0.0253), and 3.71 ± 0.41% (<0.0001), respectively, of total purines in dialysates. The total product formation in dialysate was significantly greater than the total eATP product in beaker. There was no significant difference in product formation in dialysates from nondistended and distended preparations, p > 0.05  in the suburothelium using a MD probe inserted between urothelium and detrusor and examined whether an eATP decrease and product increase can be detected in dialysate . eATP wp > 0.05 , One-wayn = 12 in each group). Thus, at 60 min of reaction, eATP was 93.63 ± 0.61% in beaker, 82.81 ± 1.93% in solutions from nondistended preparations (P = 0.4125) and 58.29 ± 7.09% in solutions from distended preparations (p < 0.0001), 2way ANOVA with Tukey’s multiple comparisons test. The decrease of eATP was greater in solutions from distended than from nondistended preparations at 10–60 min (p < 0.05), 2way ANOVA with Sidak’s multiple comparisons test. Likewise, the formation of eADP at 6–60 min, of eAMP at 30–60 min, and of eADO at 60 min was significantly greater (p < 0.05) in solutions collected from distended than from nondistended detrusor-free preparations.To determine whether soluble enzymes contribute to distention-dependent hydrolysis of eATP and eADP in the LP, the substrate was added to large-volume EL solutions that were transferred from chambers containing nondistended or distended bladders to empty chambers. The final volume of ELS plus substrate was 2.5 ml. Substrate decrease and product increase was measured for 1 h following addition of substrate. As shown in P = 0.0406) and 60 min (P = 0.0044) of enzymatic reaction in ELS from distended but not from nondistended preparations . The difference of eADP decrease in ELS from nondistended and distended bladders was significant at 40 min (P = 0.0486) and 60 min (P = 0.0197) of reaction, 2way ANOVA with Sidak’s multiple comparisons test.When eADP was used as substrate in 2.5 ml ELS , a signip < 0.05) as compared with eATP in beaker. In cELS from distended preparations, however, a significant decrease of eATP (p < 0.05) was observed at all time points except at 10 s . The decrease of eATP was significantly greater in cELS from distended than from nondistended preparations at 4–60 min of reaction (p < 0.05), 2way ANOVA Sidak’s multiple comparisons test. The formation of eADP and eAMP from eATP was significantly greater in cELS from distended than from nondistended preparations at 4–60 min and 6–60 min, respectively (p < 0.05 and 2way ANOVA Sidak’s multiple comparisons test). The increase in eADO was significant at 30–60 min (p < 0.05) of reaction in cELS from distended preparations, but it did not reach significance at all time points in cELS from nondistended preparations, 2way ANOVA Tukey’s multiple comparisons test  from both nondistended and distended preparations . In cELSons test .p < 0.0001), n = 4, 2way ANOVA, and Sidak’s multiple comparisons tests. The eADP decrease was statistically significant at 20–60 min. Significant increase of eAMP and eADO was observed at 6–60 min and 30–60 min, respectively (p < 0.0001 at 60 min for both products), n = 4, 2way ANOVA Sidak’s multiple comparisons tests  of reaction . The decrease of eATP remained unchecked in mKBS-A that contained normal Ca2+, but lacked Mg2+(n = 3) (2+ but lacked Ca2+ (n = 5), the decrease of eATP was significantly enhanced at 8–60 min of reaction when compared with KBS controls . Removal of Mg2+ and Ca2+ in the presence of EGTA (mKBS-C and n = 4) almost completely inhibited the eATP decrease (n = 3), In control ELS (KBS) from distended preparations , the sub+(n = 3) . Howevercontrols . Completdecrease . eATP hy2+ alone, was enhanced in the absence of Ca2+ plus EGTA, and was abolished in the absence of both Ca2+ and Mg2+ and addition of EDTA . As with the other e-products, the formation of eADO from eATP was abolished when both Ca2+ and Mg2+ were removed or EDTA was added   .n = 5, p < 0.0001) of reaction. The decrease of eADP was accompanied with an increase of eAMP from 7.01 ± 0.78% in beaker to 20.99 ± 2.39% at 60 min (p < 0.0001) of reaction (p < 0.0001)  did not affect significantly the degradation of eADP. In contrast to eATP, however, removal of Ca2+ and addition of EGTA  significantly inhibited the decrease of eADP substrate (2+-free solution (mKBS-A); however, this effect did not reach statistical significance for the duration of 1 h. No formation of eAMP from eADP was observed in Ca2+-free solutions containing EGTA . However, in cELS from distended preparations  . Similarubstrate . The foring EGTA . The forned EGTA .n = 9) and 46.87 ± 11.35% in the presence of ARL67156 (n = 5), P = 0.0076. eADP increase was not affected by ARL67156 at all time points of reaction , significantly diminished the decrease of eATP from 6 to 60 min of reaction , suggestENPP1 Inhibitor C diminished the decrease of eATP, did not affect the increase of eADP, and significantly reduced the formation of eAMP and eADO , suggestThe TNAP inhibitor L-p-BT had no significant effect on the eATP decrease or the eADP, eAMP, and eADO increase , suggesthttp://www.informatics.jax.org/expression.shtml) and The Human Protein Atlas (https://www.proteinatlas.org). Thus, tissue homogenates from skeletal muscle, liver, heart, and bronchus as well as from brain of a Nt5e−/− mouse were used as negative controls for antibody validation. No signals were detected in the negative control tissue homogenates  whereas ENTPD8 and TNAP were barely detected in both types of samples. Importantly, the relative expression of nucleotidases differed in urothelium and cELS. In the urothelium, ENPP1 was the second highly expressed protein and was significantly higher than ENTPD2 (P = 0.0003), ENTPD3 (p < 0.0001), ENPP3 (p < 0.0001), NT5E (p < 0.0001), ENTPD8 (p < 0.0001), and TNAP (p < 0.0001), 2way ANOVA with Tuckey multiple comparisons test (p < 0.0001), and did not differ from ENTPD2 (P = 0.6412), ENTPD8 (P = 0.9639), NT5E (P = 0.9431) or TNAP (P = 0.9657), 2way ANOVA with Tuckey multiple comparisons test (p < 0.0001) (P = 0.0006).The following enzymes were detected in both urothelium tissue homogenates  and cEL ons test . In the ons test . In the  0.0001)  whereas The present study reports two major findings: 1) distention of the bladder wall during filling is associated with increased metabolism of extracellular purine nucleotides in the LP, and 2) multiple soluble enzymes contribute to mechanosensitive degradation of purines in the LP, in addition to membrane-bound nucleotidases. To the best of our knowledge, mechanosensitive degradation of extracellular mediators has not been reported previously. Such mechanisms in the LP likely contribute to guaranteeing a proper ratio of excitatory-to-inhibitory purine mediators deep in the bladder wall that is necessary to maintain adequate bladder excitability during bladder filling.2, P2X3, and P2X2,3 purinergic receptors  ecto-nucleotidases process 1,N6-etheno-bridged purine nucleotides similarly to their endogenous counterparts  possible release of endogenous adenine purines remains undetected since the samples that were in contact with tissue do not undergo further etheno-derivatization. To examine the degradation of purine substrates at physiological levels, we chose a concentration  that is analogous to the concentration of purines previously determined in the same bladder model and with the same detection methodology  the terparts ; this alhodology . Monitorhodology . As antivia a MD probe inserted between the urothelium and the detrusor of ex vivo bladder preparations. Despite the extremely limited surface of the microdialysis membrane (∼0.6 mm2), the very low internal volume of the MD probe at the membrane (∼0.1 µl), and the short contact of the eATP substrate with LP (∼6 s), eATP produced eADP, eAMP, and eADO when perfused through the MD probe. Because the eATP breakdown occurred in such a small fraction of the LP for such a short contact between substrate and enzymes, distention-dependent eATP hydrolysis could not be revealed in these experiments. Importantly, however, this study provided affirmation that eATP can be degraded in the suburothelium and that results obtained in detrusor-free preparations can be extrapolated to the multilayer bladder wall.We next asked whether eATP hydrolysis occurs in the suburothelium in the presence of detrusor. Microdialysis has been instrumental in examining release of small-molecule substances in the interstitial space of brain, skeletal muscle, liver, kidney, adipose tissue, and skin , but hasEnhanced ATP degradation during filling explains why ATP represents such a small portion of the total purine pool in the LP at the end of bladder filling . HoweverSeveral mechanisms could underlie increased degradation of purines in preparations that were distended by filling with physiological solution. One possibility is that biaxial stretch of the urothelium during bladder filling is accompanied by increase of surface area, which results in better access of substrate to membrane-bound enzymes. Another possibility is that stretch of cells in the urothelium transduce the mechanical signal into a cascade of biochemical signals resulting in actin cytoskeleton rearrangement, which in turn alters cell shape and plasma membrane assembling . Althougin vitro, but the ATP hydrolysis rate by ENPP4 is negligible compared to that of ENPP1 (Four major enzyme families are involved in extracellular purine metabolism: 1) the ENTPD family (EC 3.6.1.5), 2) the ENPP family , 3) Ecto-5-nucleotidase NT5E/CD73 (EC 3.1.3.5), and 4) Alkaline phosphatases (EC 3.1.3.1). . ENTPD1,of ENPP1 . Purine of ENPP1 . Mammaliof ENPP1 . In factof ENPP1 . It is pof ENPP1 . In summvia equilibrative nucleoside transporters and re-phosphorylation to AMP inside the cell.The information about distribution, localization and function of nucleotidases in the non-disease bladder is rather limited. Immunohistochemistry studies of the mouse urinary bladder  reportedMembers of the nucleotidase families demonstrate relative substrate specificity for adenine purines. ENTPD1, 3, and 8 hydrolyze both ATP and ADP; however, the hydrolysis of ATP is more rapid than the hydrolysis of ADP. ENTPD1 hydrolyzes ATP directly to AMP with minimal formation of ADP as an intermediate product . The appWhile membrane-bound forms of nucleotidases have been studied extensively, soluble forms of nucleotidases have received rather little attention. Some amounts of ENTPDs, ENPPs, CD73, and alkaline phosphatase appear to constitutively circulate in human bloodstream and their levels increase in disease states such as inflammation and cancer . Neurona2+ and Ca2+ for maximal activity (WEBMAXC EXTENDED (ucdavis.edu)]  whereas in the absence of Ca2+, ∼12% of Mg2+ is chelated by 5 mM EGTA. In contrast, 5 mM EDTA chelates ∼99.99% of both cations under the same conditions. We took advantage of the different chelating properties of EGTA and EDTA and measured the degradation of eATP and eADP in solutions that contained released enzymes but lacked Ca2+ and/or Mg2+ with or without addition of EGTA or EDTA. As anticipated, removal of both Ca2+ and Mg2+ abolished the hydrolysis of eATP and eADP, indicating that Ca2+ and Mg2+ are crucial for optimal activities of soluble “ATPases” and “ADPases” in the LP. The presence of Ca2+ alone or Mg2+ alone appeared to be sufficient for the ability of soluble “ATPases” to hydrolyze ATP. Surprisingly, the hydrolysis of eATP was enhanced in the presence of Mg2+ and absence of Ca2+. It is possible that Ca2+ and Mg2+ “compete” for maintaining optimal activity of soluble enzymes so that when Ca2+ is removed, Mg2+ takes over the maintenance unrestrained. The formation of eADP from eATP followed the relationships described for the eATP decrease: no change in the absence of Mg2+ alone, enhanced formation of eADP in the absence of Ca2+ plus EGTA, and abolished eADP formation when both Ca2+ and Mg2+ were absent. Despite increased formation of eADP in the absence of Ca2+, neither the formation of eAMP nor of eADO were significantly enhanced. In fact, the hydrolysis of eADP was modestly inhibited in the absence of Mg2+ and significantly inhibited in the absence of Ca2+, suggesting that soluble ADPase(s) requires Ca2+ more than Mg2+ for its activity. This is in contrast with what we observed with the eATP hydrolysis. Altogether, the present study establishes that Ca2+ and Mg2+ are essential for the activities of soluble nucleotidases that are released in the LP during bladder filling. However, releasable enzymes that degrade sequentially ATP to ADO appear to depend unequally on extracellular Ca2+ and Mg2+: thus, Mg2+ might compete with Ca2+ for ensuring optimal activity of enzymes that convert ATP to ADP, whereas Ca2+ may be more important for the activity of nucleotidases that catabolize ADP to AMP and then to ADO.All plasma membrane nucleotidases require millimolar concentrations of divalent cations Mgactivity . As partis.edu)] , 5 mM EGUniProt). In ELS collected from distended preparations, PSB06126 diminished the eATP decrease and the eADP increase and had no effect on eAMP and eADO increase. The ENPP1 Inhibitor C , suggesting that no significant differences in the mass of membrane-bound and cleaved enzymes would be detected with western immunoblot methodologies. This might explain the similar masses (molecular weights) of enzymes that are present in urothelial tissue homogenates and in cELS. Studies have suggested that lack of transmembrane domains of nucleotidases may result in reduced catalytic activity  multiple enzymes that coexist on the cell surface or are released during bladder filling determine the effective concentrations of biologically active mediators in the LP and the degree of activation of purinergic receptors in various types of cells in the bladder wall, and 2) in studies of purinergic signaling, it is important to investigate simultaneously the availability of extracellular ATP and its metabolites.In summary , here weAt present, we do not know the mechanisms underlying the distention-induced enzyme release in the LP. The mechanisms of coordination of multiple enzyme activities in regulation of bladder excitability also remain to be unraveled. Future studies aimed at understanding the complexity of purinergic regulation and functions in the bladder will help to guide translational and clinical efforts for bladder motility disorders, inflammation, cancer, and other human diseases.