The compounds crystallize in a filled version ofthe orthorhombic Zr2Nix1–P-type structure with x = 0.52; their stoichiometry,determined from both single-crystal and powder X-ray diffraction data,is centered on Ho2Ni0.8T1.2 witha narrow solid solubility range for the silicide, while the germanideappears to be a line phase. In addition to R = Dy and Ho, R2Ni0.8T1.2 compounds also form for R = Y andTb, representing the first examples of rare-earth-based compoundsadopting the Zr2Nix1–P structural prototype. Bulk magnetization data reveal the main transitionsof the ferrimagnetic or ferromagnetic type at TC = 38 K for Ho2Ni0.8Si1.2 and TC = 37 K for Ho2Ni0.8Ge1.2, which are followed by subsequent magneticreordering at lower temperatures. Neutron diffraction shows complexmagnetic structures below TC with bothferromagnetic and antiferromagnetic components and magnetic propagationvector κ1 = . Below TN ≅ 24 K (22 K) for the silicide (germanide), an additionalantiferromagnetic coupling following an incommensurate magnetic propagationvector κ2 =  appears to coexist with the first magnetic structure.We report two newrare-earth (R) ternary intermetallic compounds—Ho 2Ni0.8Si1.2 (and Ho2Ni0.8Ge1.2) [Zr2Ni0.48P-type (oP32-Pnma)] viewed along the b axis is given. The Ho, Ni1,Ni2/Si, Si1 and Si2 atoms are represented as red, turquoise, blue,light-gray, and dark-gray spheres, respectively. The distinctive structuralfragment is built up of five differently oriented trigonal prisms.The magnetic structure of Ho2Ni0.8Ge1.2 with the antiferromagnetic component in the c directionand the ferromagnetic component along the a axis.The crystal structure of Ho Among many classes of different materials, intermetalliccompounds represent a vast and excellent resource, many of them witha strong potential to be deployed in various technological applications.Of note, R-based compounds, where R = rare earth, remain among themost interesting and investigated because of the emergence of unique,sometimes exotic, properties and functionalities brought about bythe R atoms and their peculiar electronic structures, which also makesthem crucial materials for many technologies.2 and Ho–Ni–Ge3 systems, we identified intermetallics formingwith theapproximate compositions of R5Ni2T3, where T = Si or Ge. At the time, the crystal structures, exactcompositions, and properties of these two new compounds remained unknown.More recently, the same phase has been reported to form also in theHo–Ni–Si ternary system, however, still without an investigationand determination of its crystal structure.4During our earlier exploration of new ternary phases in theDy–Ni–Si5Ni2T3” compounds for T = Si andGe and found them to crystallize in an orthorhombic unit cell whoseprototype is Zr2Nix1–P .5 Therefore,the true stoichiometry is not R5Ni2T3 but very close to it, namely, R2Nix1–Tx1+, with 0.094(1) ≤ x ≤ 0.250(1) for Ho2Nix1–Six1+ and 0.190(1) ≤ x ≤ 0.201(1) for Ho2Nix1–Gex1+. Noting that noother rare-earth–transition-metal silicides or germanides haveever been reported to crystallize with the Zr2Nix1–P prototype,9 we prepared homologues with other heavy lanthanides,finding that the phase with R = Gd does not form, while it forms whenR = Y, Tb, Dy, and Ho. In addition to the formation and crystal structureof these new R2Nix1–Tx1+ ternary intermetallic compounds,we report physical properties and magnetic structures of the two Hocompounds at the Ho2Ni0.8T1.2 compositionfor T = Si and Ge.In this work, we establish the crystal structures of the “Ho22.110 The purities of non-rare-earth elements purchasedcommercially were 99.99 wt % for Ni and 99.999 wt % for Si and Ge.The total mass of the samples was 3–4 g for crystallographicinvestigation and physical property measurements and 7–8 gfor neutron diffraction investigation. The buttons were melted threetimes, turning them upside down after each melting to ensure homogenization.Weight losses were less than 0.7 wt % (less than 0.3 wt % for thesamples for neutron diffraction). Particular care was taken duringthe melting and cooling processes  because the alloys are sensitive to thermal shock, with a strongtendency to shatter into pieces. Initially, samples for the crystalstructure determination, for phase analysis, and for the checkingof limits of solid solubility were prepared for R = Ho with threenominal compositions: Ho50Ni18.3T31.7, Ho50Ni20T30, and Ho50Ni25T25 for both T = Si and Ge. Later, sampleswith the nominal compositions Ho50Ni20T30 (T = Si and Ge) were prepared for neutron diffraction investigation.The as-prepared ingots were wrapped in a Ta foil and sealed undervacuum in a quartz tube. They were annealed at 1000 °C for 4–7days followed by air cooling to room temperature after the ampouleswere taken out of the furnace.Polycrystalline samples  were prepared by arc melting from the constituent elements,weighed in stoichiometric amounts, under a pure TiZr-gettered Ar atmosphere.The purity of the rare-earth metals was 99.9+ wt % with respect toall other elements in the periodic table .Aztec software)]. EDX analyseswere performed on at least four sample points (or areas) to identifythe phase composition, with a counting time of 60 s. Extra phasespresent as impurities were used as internal standards; the precision of the measurementswas estimated to be within 1 atom %. SEM images of the samples weretaken using both backscattered and secondary-electron modes.The microstructure and homogeneityof the alloys were checked by light optical and scanning electron(SEM) microscopies, with the latter performed on an instrument equippedwith an energy-dispersive X-ray (EDX) microprobe for semiquantitativeelemental analysis were used to collect the powder X-ray diffraction (XRD) data; theGuinier patterns were indexed with LAZY PULVERIX,11 and accurate lattice parameters were obtainedby least-squares refinement. The Rietveld refinements were carriedout by using the FullProf program.12 The single-crystal XRD was performed at room temperatureon either a Bruker Apex CCD diffractometer or a Bruker D8 Venturediffractometer (both with Mo Kα radiation), utilizing the APEX2 and APEX3 software suites  for data collectionbetween 2 and 60° of 2θ, integration, polarization, andempirical absorption correction.14 The SHELXTL suite and XPREP algorithms wereused to check for extinction conditions and E statisticsin the intensity data sets necessary for assignment of the proper Pnma space group. Direct methods were used for structuresolution (SHELXS-97).15APEX3 software was then used to carry out fullstructure refinement .A PANanalytical X’Pert diffractometer  was performed by using a Netzsch 404 thermal analyzeron bulk samples of 0.7–0.9 g, either as-cast or annealed, sealedin an outgassed Mo crucible under an Ar atmosphere. Data were recordedupon heating at 20 K/min and upon cooling at 10 K/min (temperaturemeasurement accuracy ±5 K). The results obtained from DTA were,however, inconclusive for both Ho2.32Nix1–Si1+x and Ho2Nix1–Gex1+ phases; these turned outto be single phases with compositions close to Ho2Ni0.8Si1.2 and Ho2Ni0.8Ge1.2 . The measurements wereperformed on the samples prepared in the middle of the solid solubilityrange of the two Ho2.4T = 1.2 K intervals. High-resolution data were taken on D2B at 300K for both compounds using the additional 10′ collimation ofthe primary beam. Data analysis was performed using the Rietveld refinementprogram FullProf;12 magneticsymmetry analysis was performed using the program BASIREPS.18The neutron diffraction investigationswere performed at the ILL,Grenoble, France, using the high-resolution powder diffractometerD2B (λ = 1.594 Å) and the high-intensity powder diffractometerD1B (λ = 2.52 Å). The temperature dependencies of the powderneutron diffraction patterns (thermodiffractograms) were measuredon D1B between 1.5 and 40 K for the Si compound and between 1.5 and46 K for the Ge compound by taking data at Δ33.150Ni20Si30.We found that the structural prototype of this and all other new compoundsis the orthorhombic Zr2Nix1–P .5 The prototypical phosphide is Ni-deficient, formingat x = 0.52, which yields a composition Zr2Ni0.48P. The Zr2Ni0.48P structurefeatures eight independent 4c Wyckoff sites, fourof which are occupied by the largest Zr atoms, two by the Ni atoms,and two by the smallest P atoms. Both of the Ni sites are partiallyoccupied, at 19% and 76%. Unlike in the Zr2Ni0.48P prototype, all of the 4c Wyckoff sites are fullyoccupied in Ho50Ni20T30 when T =Si or Ge, with one of the four non-rare-earth atom sites clearly showingmixed occupancy by Ni and Si and another possibly a minor mixing ofNi of the site predominantly occupied by Si. The final refined compositionfor T = Si is Ho50Ni19.2(1)Si30.8(1), which corresponds to the Ho2Ni0.769(5)Si1.231(5) formula unit, hence suggesting that the correct stoichiometryof these new silicides and germanides, previously reported as R5Ni2T3, should be R2Nix1–Tx1+. The details of single-crystal analysis for the Ho2Ni0.769(5)Si1.231(5) compound are shown in Table S1, the refined atomic coordinates, siteoccupancies, and isotropic displacement parameters are listed in Table S2. As in the prototype, the unit cell accommodates eight 4c Wyckoff positions: four of them are fully occupied bythe larger Ho atoms and four by the smaller Ni and Si atoms, includingone site populated by a statistical mixture of Ni and Si in nearlyequal ratio (52% Ni + 48% Si) and another that is mostly populatedby Si atoms (98% Si + 2% Ni). Consequently, and for the sake of simplicity,in the following discussion, full Si occupancy is assumed for theSi1 Ni18(1)Si31(1), Ho50(1)Ni20(1)Si30(1) , and Ho50(1)Ni22(1)Si28(1), respectively. The results of microprobe analysis, therefore, indicatethat the Ho2Nix1–Six1+ compound is stable over a certain rangeof x.SEM images, representativeof the microstructure of the sampleswith starting nominal compositions HoThe Rietveld refinements of the powderXRD patterns of all preparedsamples with R = Ho are shown in Tables S3–S8. The nominal compositions,the resulting compositions of the main Ho2Nix1–Tx1+ phase from EDX analyses,and the Rietveld-refined stoichiometries along with the lattice parameters,observed unit cell volumes (Vobs), andvolume contractions (ΔV), are collected in V % = [/Vcalc] × 100, where Vcalc is the cell volume calculated from the individual atomic volumes19 and Vobs is theexperimentally observed cell volume. Formation of the Ho2Nix1–Tx1+ phases is accompanied by 10–12% volume contraction,indicating a high thermodynamic stability of these compounds, whichexplains the high melting/decomposition temperatures .Crystallographic data obtained from the Rietveld refinements, bothfor the main phase and for the detected impurity phase(s), are collectedin x ≤ 0.250(1) (Δx ≈ 0.16) for Ho2Nix1–Six1+ and 0.190(1) ≤ x ≤ 0.201(1) (Δx ≈0.01) for Ho2Nix1–Gex1+. The much narrower and practically negligiblesolubility range of the germanide phase compared to that of the silicidephase is likely due to the larger atomic size of Ge compared to thatof Si .19Rietveld refinements confirm the bulk crystal structure determinedusing a small single crystal. Further, powder XRD data indicate thatthe two new orthorhombic phases exist over limited ranges of concentrations,that is, 0.094(1) ≤ 2Nix1–Tx1+ compounds belong to an extendedfamilyof rare-earth intermetallics, the crystal structures of which canbe described by the packing of trigonal prisms formed by R atoms coordinatingboth M and T atoms . Here, these prisms arelinked together by sharing square and triangular faces, forming acharacteristic structural motif of interconnected columns, consistingof five trigonal prisms in a cross section, infinitely extending alongthe short b-axis direction ] and HoSi  compounds;9 both structure typesare based on the trigonal-prismatic coordination of the Ni and T atoms(2Nix1–Tx1+ is such that (i) the numberof heteroatomic bonds (Ni–T) is maximized and (ii) the numberof homoatomic T–T contacts is minimized .This result is quite interesting because it suggests a strong bondinginteraction between the Ni and T atoms, and because a stoichiometricT/Ni ratio of about 1.5 should instead favor the formation of T–Tbonds. In 2Ni0.769(5)Si1.231(5) is also compared with that of Ho3NiSi2 (Gd3NiSi2-type). Both compounds show a similar arrangementof structural fragments, with characteristic trigonal-prismatic coordinationand a similar orientation of the respective structural motifs. Allof the Ho atoms show the same coordination number CN = 17, and theircoordination polyhedra are very similar, corresponding to irregularpentagonal prisms capped on all of the faces.The trigonal prisms may be distorted depending on the kind of atomhosted inside them; i.e., the prism edges are longer (shorter) whenthe prism coordinates the larger  Si (Ni) atoms. These distortionsare similar to those observed in the binary HoNi 21 with latticeparameters a = 11.2922(1) Å, b = 4.1542(1) Å, and c = 11.1410(1) Å.We also found that this new R3.23.2.1M(T), have been measured between 2 and 300 K in applied magnetic fieldsof 200 Oe, 500 Oe, and 5 kOe for Ho2Ni0.8Si1.2  data reveal main transitions thatare either ferrimagnetic (FIM) or ferromagnetic (FM) in nature, occurringat TC = 38 K for the silicide and TC = 37 K for the germanide. These transitiontemperatures are determined from the minima of the first derivativesof the magnetization with respect to the temperature , it is reasonable to assume that the additional low-temperatureanomalies are intrinsic to these compounds. Thermomagnetic irreversibilitiespresent between the ZFC and FC data below about 10–20 K areconsistent with the nonzero hysteresis developing at the lowest temperature.The magnetic behaviors of these two compounds are similar. The H/M, is shown in parts a and b of T) = C/(T – θP) (with C being the Curie constant) above 100 Kfor the silicide and above 75 K for the germanide. The least-squaresfits to the data in the paramagnetic region give an effective magneticmoment, peff, of 10.68 μB for Ho2Ni0.8Si1.2 and 10.75 μB for Ho2Ni0.8Ge1.2. Bothvalues are very close to the Hund’s rule derived theoreticalvalue of 10.61 μB for the Ho3+ ion,23 indicating that the Ni magnetic moment is quenched,as is common for many other rare-earth compounds containing Ni.25 The positive values of the Weiss temperatures, θP, of 39 and 38 K respectively for the silicide and germanide arecommensurate with either the FM or FIM ground states.The inverse magnetic susceptibility, 1/χ = M(H), measured at T = 2 K (bothcompounds) and 15 and30 K for the silicide in magnetic fields up to 70 kOe, is shown in The isothermal magnetization, T = 2 K show noticeable hysteresis,which is typical of FM/FIM materials formed by lanthanides with nonzerosingle-ion anisotropies. The M(H) data do not reach saturation even at the highest applied magneticfield of 70 kOe, approaching 8.33 μB/Ho atom, a valuelower than the expected 10 μB/Ho if all Ho magneticmoments would align ferromagnetically, possibly indicating antiferromagnetic(AFM) contributions rather than a simple collinear FM ordering. Acomparison between the isothermal magnetization of the two compounds,for data measured at 2 K, is shown in Figure S2; a very similar behavior is observed, with the coercive field slightlylarger for the silicide .The measurements at 3.2.2The heat capacityhas been measured for both compounds between 2 and 100 K in zero andapplied magnetic fields of 10, 20, and 30 kOe 9a,b.M(T) data, indicating that these phasechanges are second-order. Plotting CP/T versus T of the zero-field data (insetsof M(T) data.Rapid upturns observed below ∼4 K in both compounds reflectthe hyperfine field contributions commonly observed in other Ho-basedintermetallics.26 Under the applied magneticfield, the main peaks initially move to lower temperatures, suggestingat least some degree of antiparallel coupling between the Ho magneticmoments in both compounds. This supports the idea mentioned abovethat the low value of the magnetization at 70 kOe and its nonsaturationmight be caused by AFM contributions. Broad anomalies are also observedat about 12 K for both compounds, likely indicating spin reorderingunder a magnetic field.The data show the main λ-type peaks in agreement with theglobal magnetic ordering transition temperatures determined from the 3.32Ni0.8Si1.2 and Ho2Ni0.8Ge1.2 compounds as well as themixed Ni/Si occupation of only one of the eight 4c Wyckoff sites (2Ni0.78(1)Si1.22(1) and Ho2Ni0.76(2)Ge1.24(2). No sign of the presenceof any visible amounts of impurity phases is found.High-resolutionpowder neutron diffraction data confirm the orthorhombic crystal structureof both the Hoff sites 10a,b, wiTC = 38 and 37 K for thesilicide and germanide, respectively.The thermodiffractogramsof the two compounds as measured using high-intensity powder neutrondiffraction are shown in K-search, which is part of the FullProf suite of programs12 with a magnetic propagationvector κ1 = . Figure S3 shows the integrated intensity of the strongest among allof the magnetic peaks  of thisκ1 phase (the first magnetic phase appearing uponcooling), as a function of the temperature, for Ho2Ni0.8Ge1.2 and Ho2Ni0.8Si1.2 . A second magnetic transition seems to take placeat about TN = 24 K in Ho2Ni0.8Si1.2 and at about 22 K in Ho2Ni0.8Ge1.2, where a single new magnetic Bragg peakat very low 2θ values appears in both compounds. We will firstdiscuss the magnetic structure present below TC before we deal with the situation below TN. Magnetic symmetry analysis using the program BASIREPS.All magnetic Bragg peaks can be indexed using the program  BASIREPS,18 detera direction and an AFM coupling (BV) inthe c direction  allows refinement of the diffraction data below TC. In order to have an increased sensitivity to the magneticdiffraction intensity, we refined difference data sets created bysubtracting the purely nuclear intensity recorded in the paramagneticregion above TC. Among the eight allowed IRs, only one having a FM BV alongtheunit cell 2Ni0.8Ge1.2 in c direction of Ho1,Ho2, and Ho4 that are antiferromagnetically aligned along the a direction, separated by an AFM strip of Ho3 spins, whichextends as well along the c-axis direction , this is not obvious, as has been shown,for example, in the intermetallics Ho5Ni2In4 or Ho11Ni4In9, where differentmagnetic propagation vectors and different temperature dependencieshave been found even for Ho ions occupying the same Wyckoff site.252Ni0.8Ge1.2 at 26.6 K and for Ho2Ni0.8Si1.2 at 24.5 K the values of the AFM component along the c direction, of the FM component along the a direction,and of the resulting total magnetic moment for all of the Ho atoms.As theFM component along the lections 11, they TN. The closeness of the magnetic transition temperature TN ∼ 24 K of Ho5Si322—a possible impurityphase—to the here determined TN = 24 K for the silicide compound and a certain similitude of themain magnetic Bragg peak could question the origin of this secondtransition as coming from the Ho2Ni0.8Si1.2 sample. The absence of any visible impurity phase in thehigh-resolution and high-intensity neutron diffraction data and thestrong intensity of the additional low-angle magnetic peak exclude,however, this possibility. The same arguments speak against the originof the low-angle magnetic peak in Ho2Ni0.8Ge1.2 as coming from a hypothetical Ho5Ge3 impurity; furthermore, in this case the transition temperaturesare significantly different because TN = 27 K for Ho5Ge322 and TN = 22 K for Ho2Ni0.8Ge1.2.As was already indicated above, the appearance ofa very strongadditional magnetic Bragg peak at very low 2θ values indicatesa change in the magnetic structure and can be looked upon as a secondmagnetic transition defining a Néel temperature 2 =  for Ho2Ni0.8Si1.2 and κ2 =  for Ho2Ni0.8Ge1.2. Magneticsymmetry analysis proposes four IRs, with two representing sine wavesand two others representing cycloidal types of magnetic structures.Only one sine-wave model is able to reproduce the magnetic peak andthe absence of any further magnetic peaks with reasonable magneticmoment values (b direction with a maximum amplitudeof 5.0(1) μB at 1.5 K for Ho2Ni0.8Ge1.2. Here, all Ho sites were constrained to have thesame amplitude and the same phase. The superposition of this κ2 =  modulationwith the κ1 =  order leads to a magneticstructure sketched in The fact that only one broad newmagnetic peak appears makes thedetermination of a magnetic propagation vector ambiguous, becausean unlimited number of incommensurate values can be found that reproducethe position of the peak. Limiting oneself to magnetic propagationvectors having only one component and using the information on theabsence of further new magnetic peaks, a simple solution can be foundwhere κt values 12b. This1 order and of thesine wave at 1.5 K, together with the resulting total magnetic momentsof each site. The refinement assumed hereby that both magnetic couplingsextend over the whole sample volume. A phase segregation scenariowhere one part of the sample volume follows κ1 andthe second part κ2 cannot be taken into considerationas a solution because the total moment values would be higher thanthe free ion value of Ho3+, which is μ3+Ho = 10.61 μB. The fact that the temperaturedependence of the magnetic peaks created at TC do not show any change at TN  short-rangeorder of the κ2 type appears as well already. Nofurther clear peak in the CP data is thereforecreated at TN because only the correlationlength of the coupling and the value κx of the magnetic propagation vector κ2 arechanging. Figure S4 shows, furthermore,that a small anomaly is visible at around 13–14 K in the temperaturedependence of the intensity of the strong low-angle peak of the κ2 phase as well as in its full width at half-maximum and itsposition. It indicates a small change in the details of this incommensuratemagnetic structure and can be related to the small anomaly seen inthe magnetization data at 13 K.Comparingthe results of neutron diffraction with those of theheat capacity and magnetic data, the question remains, why is thesecond transition at 42Ni0.8T1.2 with T= Si and Ge, are reported.They correspond to the unidentified phase R50Ni20T30, namely, “Ho5Ni2T3”, earlier reported to form in the ternary systemsDy–Ni–T and Ho–Ni–T and crystallize witha filled version of the orthorhombic unit cell of the Zr2Nix1–P type .While this prototype presents a vacancy of x = 0.52,which translates into a resulting stoichiometry of Zr2Ni0.48P, the stoichiometry of the two Ho compounds studied hereis centered on Ho2Ni0.8T1.2 witha very narrow solid solubility range for the silicide, while the germanideturns out to be a line phase. In addition to R = Dy and Ho, R2Ni0.8T1.2 compounds are also formedfor R = Y and Tb; attempts to prepare the homologous Gd-based Gd2Ni0.8T1.2 failed. The R2Ni0.8T1.2 compounds constitute the first example ofan R-based compound crystallizing with the Zr2Nix1–P type, as well as the first case of an intermetallicphase adopting this ternary structural prototype.The crystal structure, magnetic properties, and magnetic structuresof two new rare-earth-based intermetallic compounds, Ho2Ni0.8Si1.2 and 37 K for Ho2Ni0.8Ge1.2, isrevealed by the magnetization data; this main transitionis then followed by subsequent magnetic orderings at lower temperatures.The susceptibility data in the paramagnetic region give effectivemoments, μeff, of 10.68 and 10.75 μB for the silicide and germanide, respectively; both values are veryclose to the theoretical value of 10.61 μB. Neutrondiffraction shows the existence of two magnetic propagation vectorsin both compounds. First transitions at TC = 38 K for the silicide and at 37 K for the germanide lead to acommensurate κ1 =  magnetic structure havingFM and AFM components. At lower temperatures, an additional AFM coupling,appearing below TN2 ∼ 24 K forthe silicide and ∼22 K for the germanide and following an incommensuratemagnetic propagation vector κ2 = , coexists with the first magnetic structure.FIM- or FM-typeordering, at 38 K for Ho


RyR1 channel activity is modulated by ligands, including the activators Ca2+ and ATP. Patients with inherited mutations in RyR1 may exhibit muscle weakness as part of a heterogeneous, complex disorder known as RYR1-related myopathy (RYR1-RM) or more recently termed RYR1-related disorders (RYR1-RD). Guided by high-resolution structures of skeletal muscle RyR1, obtained using cryogenic electron microscopy, we introduced mutations into putative Ca2+ and ATP binding sites and studied the function of the resulting mutant channels. These mutations confirmed the functional significance of the Ca2+ and ATP binding sites identified by structural studies based on the effects on channel regulation. Under normal conditions, Ca2+ activates RyR1 at low concentrations (µM) and inhibits it at high concentrations (mM). Mutations in the Ca2+-binding site impaired both activating and inhibitory regulation of the channel, suggesting a single site for both high and low affinity Ca2+-dependent regulation of RyR1 function. Mutation of residues that interact with the adenine ring of ATP abrogated ATP binding to the channel, whereas mutating residues that interact with the triphosphate tail only affected the degree of activation. In addition, patients with mutations at the Ca2+ or ATP binding sites suffer from muscle weakness, therefore impaired RyR1 channel regulation by either Ca2+ or ATP may contribute to the pathophysiology of RYR1-RM in some patients.The type 1 ryanodine receptor (RyR1) is an intracellular calcium (CaThe online version contains supplementary material available at 10.1186/s40478-021-01287-3. Using the high-resolution RyR1 structure solved in our laboratory we identified binding sites for the channel activators Ca2+ and ATP. Mutagenesis of these sites combined with functional studies confirmed that they are indeed the key ligand binding sites. Both the ATP and Ca2+ sites are involved in disease-causing mutations that alter the response of the channel to these physiological activators and likely contribute to the pathophysiology of RyR1-RD.The ryanodine receptor/calcium release channel (RyR1) is required for skeletal muscle excitation–contraction (EC) coupling. Mutations in RyR1 that render the channel leaky (unable to close properly) to calcium  coupling in skeletal muscle. RyR1 is a homotetramer comprised of four 565 kDa protomers and as such is the largest known ion channel. In addition, regulatory and targeting proteins for enzymes including protein kinase A (PKA) and CaM kinase II (CAMKII), are associated with the channel and regulate its function , 45.RYR1-related myopathies (RYR1-RM), or as recently proposed RYR1-related disorders (RyR1-RD) -ATP binding, [3H]-ryanodine binding, which is a surrogate measure of channel activity, and single channel recordings in planar lipid bilayers were performed. The RyR1 T4979F mutant channel exhibited reduced [α-32P]-ATP binding, with a Bmax of 2.5 ± 0.25 pmol ATP/pmol RyR1 compared to a Bmax of 8.0 ± 0.77 pmol ATP/pmol RyR1 for WT RyR1. These data confirm that T4979 is a critical residue for ATP binding to RyR1. Furthermore, the Bmax of 8 suggests that there is likely a second ATP binding site in RyR1 with two molecules of ATP are binding per protomer of the homotetrameric channel. In contrast, the K4211S/K4214S/R4215S mutant channel exhibited normal [α-32P]-ATP binding to RyR1 , near the adenine ring of ATP, and a triple mutant of K4211/K4214/R4215, each to serine, near the triphosphate tail of ATP was generated. These mutants were made in recombinant rabbit RyR1 and expressed in HEK293 cells. To assess the effects of these mutations, [α-3H]-ryanodine binding to further evaluate activation of RyR1 by ATP binding. Ryanodine binds to pore of the RyR1 channel in the open state [3H]-ryanodine binding to T4979F RyR1 and K4211S/K4214S/R4215S RyR1 were similar to that of the WT RyR1 , the open probability was ~ 90% [2+]cyt. Mutant RyR1 channels T4979F and K4211S/K4214S/R4215S exhibited similar single channel properties compared to WT RyR1 under this condition, suggesting normal Ca2+ dependent activation. Addition of 1 mM ATP dramatically increased WT RyR1 Po  and S4028L. The Q3970K mutation is linked to a form of RYR1-RD formerly referred to as multi-minicore disease [2+ dependent activation measured using single channel determinations and [3H]-ryanodine binding  prevents the entry of ATP into that site, thus explaining why ATP binding to the T4979F mutant RyR1 is reduced. Other adenine nucleotides such as ADP, AMP, or adenosine can also increase the Po of RyR1, but with reduced efficacy cyt in resting cells is approximately 100–150 nM and rises to at least 1 µM following Ca2+ release through RyR1 during EC coupling. Mutations of RyR1 residues E3893 and E3967 to either A or D significantly reduced the high affinity Ca2+-dependent-activation of RyR1 compared to the WT channel , this Ca2+ binding site was occupied by one Ca2+, which forms a brige between the CTD and CSol to stabilize the open state of the RyR1 channel, whereas at high Ca2+ concentration (mM), CTD and CSol each bind to one Ca2+, which disrupts the CTD-CSol interface to make the channel close. A patient with a Q3970K mutation at this site exhibited the same impaired Ca2+-dependent activation and deactivation as the E3893A/D and E3967A/D mutant channels, which is consistent with a previous study showing that RyR1-Q3970K displayed low Ca2+ dependent channel activity cyt, which is consistent with channel leak -ryanodine or 5 nM [α-32P]-ATP, 1 M NaCl, 20 mM HEPES, and 0.5 mM EGTA at 37 °C for 2 h. The concentration of free Ca2+ was calculated with WinMaxC . For ATP activation, 1 mM ATP and 1 mM free Ca2+ were added during incubation. The binding mix was then filtered through Whatman GF/B filters presoaked with 1% polyethyleneimine. The filters were washed three times with 5 mL of ice-cold washing buffer containing 0.2 M NaCl and 5 mM HEPES (pH 7.5) to remove unbound [3H]-ryanodine, and the amount of remaining [3H]-ryanodine was determined by liquid scintillation counting. Nonspecific binding was determined by measuring [3H]-ryanodine binding in the presence of 10 μM unlabeled ryanodine. All binding assays were done in duplicate.Skeletal muscle SR microsomes or ER vesicles from HEK293 cells expressing WT or mutant RyR1 were incubated in media containing 5 nM -ryanodine binding at the indicated [Ca2+]cyt. Data are presented as mean ± S.E.M from 4 for each group *P < 0.05 vs. WT; #P < 0.05 vs. RyR1-S4028L, ANOVA, Tukey-Kramer with post hoc correction.


COL4A3, COL4A4, or COL4A5 gene. The knockout mice for Col4α3, Col4α4, and Col4α5 are developed and well characterized for the study of Alport syndrome. However, disease progression and effects of pharmacological therapy depend on the genetic variability. This model was reliable only to mouse. In this study, we created a novel Alport syndrome rat model utilizing the rGONAD technology, which generated rat with a deletion of the Col4α5 gene. Col4α5 deficient rats showed hematuria, proteinuria, high levels of BUN, Cre, and then died at 18 to 28 weeks of age . Histological and ultrastructural analyses displayed the abnormalities including parietal cell hyperplasia, mesangial sclerosis, and interstitial fibrosis. Then, we demonstrated that α3/α4/α5 (IV) and α5/α5/α6 (IV) chains of type IV collagen disrupted in Col4α5 deficient rats. Thus, Col4α5 mutant rat is a reliable candidate for the Alport syndrome model for underlying the mechanism of kidney diseases and further identifying potential therapeutic targets for human renal diseases.Alport syndrome is an inherited chronic human kidney disease, characterized by glomerular basement membrane abnormalities. This disease is caused by mutations in  The 6 chains assemble into 3 types of heterotrimers (protomers) which have distinct tissue distribution and function. In the mammalian kidney, the glomerular basement membrane (GBM) requiring long-term maintenance of the glomerular filtration functions, is composed of α3/α4/α5 (IV) isoforms. Whereas α1/α1/α2 (IV) isoforms are found in all basement membranes, and α5/α5/α6 (IV) isoforms are detected in Bowman’s capsule (BC)3.Type IV collagen networks are the structural foundation for all basement membranes, consist of 6 different chains from COL4A1 to COL4A6 in mammals4. Alport syndrome is caused by mutations in COL4A3, COL4A4, or COL4A5 gene encoding type IV collagen α3, α4, or α5 chains5. Defects in the COL4A5 gene cause X-linked Alport syndrome, which accounts for about 80% of Alport syndrome6. And the remaining cases are associated with mutations in COL4A3 or COL4A4 genes. The pathological events of Alport syndrome are very similar, are found that lack of COL4A3, COL4A4, or COL4A5 resulted in the disorganization of α3/α4/α5 (IV) network1.Alport syndrome is a basement membrane disorder, characterized by hereditary nephropathy that results in irreversible, progressive renal failureCol4α3, Col4α4, and Col4α5 are developed and well characterized12. These model mice led important aspects of the renal disease. Whereas, there are some significant differences depend on genetic background in Alport syndrome model mice14. For example, 129X1/Sv or C57BL/6 was different patterns of disease progression in Col4α3-/- mice15. Indeed, in the human patients, the disease progression and effects of pharmacological therapy were shown to genetic variability6. However, in spite of variabilities displayed in the genetic background, only a few mammal models (only mice and dogs16) have been established for Alport syndrome.In the past decades, several Alport syndrome animal models have been produced in mouse. The mutant mice for Rattus norvegicus) is a common experimental model for the human diseases and the drug testing19. For example, Wistar Kyoto (WKY) rat strain is known to be uniquely susceptible to crescentic glomerulonephritis among the strains tested21. Injection of isologous monoclonal antibodies caused anti-glomerular basement membrane antibody-induced glomerulonephritis (anti-GBM nephritis) in WKY rats23. Although there are several advantages comparing with mice, production of genetically engineered rat models has not yet been extensively proceeded during the past decades. The recent CRISPR/Cas9 system is the simplest for generating rats carrying a modified genome25, however, the standard method for genome-editing in mammals involves 3 major steps: isolation of zygotes from females, micromanipulation ex vivo, and transfer into pseudopregnant females. These 3 steps require extremely high level of technical expertise and its proficiency of the researchers as well as technicians. To simplify these complexed and laborious processes, we established to produce knock-out and knock-in mouse and rat with high efficiency, named as i-GONAD (mouse) and rGONAD 30. The rGONAD method involves 2 key steps: an injection of the solution containing Cas9 protein, guide RNA and single strand DNA (ssDNA) into the oviduct, followed by electroporation. The rats that have undergone rGONAD are bred in routine way until birth. Moreover, rGONAD is highly efficient in both knock-out and knock-in rats30.The laboratory rat  domains of type IV collagens . Proteinuria was observed (about 6.0 mg/16 h) by 6 weeks of age in hemizygous mutant males , periodic acid Schiff (PAS), periodic acid methenamine silver (PAM), and Masson trichrome (MT)  Fig. . At 8 weMT) Fig. a. HoweveMT) Fig. a. By the31. LVSEM revealed the coarse meshwork structure of the GBM with numerous pin-holes in hemizygous mutant males, in contrast to the smoothly arranged surface in wildtype males  was performed, as described previouslyles Fig. . The trales Fig. . Moreoveles Fig. .Figure 3Col4α5 deficient rats, we examined kidney sections from wildtype and Col4α5 deficient rats stained with antibodies specific for α-smooth muscle actin (α-SMA) and fibronectin 34. To characterize such possibilities in Alport model rats, we performed immunostaining and Western blotting of Col4α5 deficient male kidneys for measuring the levels of these proteins. We first observed that an antibody to the laminin β2 chain exhibited intense staining of the GBM in Col4α5 deficient males from 8 weeks of age  and LDL receptor (LDLR) are highly expressed in renal tubules of Alport miceage Fig. a. In adding Fig. b.Figure Col4α5 deficient rats, we investigated Col4α5 deficient rats with graded levels of the other’s type IV collagen α1, α2, α3, α4, and α6 (IV). First, we produced monoclonal antibodies that specifically recognize rat COL4A6 protein. We obtained rCol4A6 antibodies by injecting the antigen in Col4a5 deficient males, but we could not obtain any specific antibody injected to wildtype rats at all. Immunoblot analysis revealed that the monoclonal anti-rCOL4A6, reacted specifically with a band corresponding to the position of the similar molecular weights in recombinant rCOL4A6 proteins, and only reacted with rCOL4A6, but not with other rCOL4 proteins  , α3/α4/α5 (IV) (GBM), and α5/α5/α6 (IV) (Bowman’s capsule) protomers were observed in the glomeruli. To examine whether localizations and/or levels of the proteins were changed in Col4α5 deficient rats changed, we performed the immunofluorescence analyses. At 8–20 weeks of age, COL4A5 expression was absent in hemizygous mutant males, and present in a mosaic pattern in heterozygous mutant females  in the kidney of les Fig. –14. The les Fig. –14. At 8ats Fig. . At 20 wCol4α5 deficient males  by Western blot analyses that were prepared from kidneys of wildtype and hemizygous mutant males Fig. . There wles Fig. . Then, tles Fig. . TherefoCol4α5 deficient rats revealed typical physiological, pathological, and also histological characteristics of Alport syndrome.The present study showed that we successfully created a novel Alport syndrome rat model with the rGONAD technology. COL4A3, COL4A4, or COL4A55. Several mouse models in Col4α3, Col4α4, and Col4α5 mutation have been known and characterized14. Col4α3-/- or Col4α4-/- mice are well used for studying treatments of Alport syndrome compared to Col4α5-/Y mice39. Because, a survival ratio of Col4α5-/Y mice was so diffusive ranged from 6 to 34 weeks10, in contrast, those of Col4α3-/- or Col4α4-/- mice were more uniform (from 13 to 26 weeks)41. In this study, Col4α5 mutant males died at 18 to 28 weeks (about 95% from 20 to 26 weeks) of age. Thus, it is useful to study experiments for prolong the lifespan of Col4α5 deficient rats for effects of the drug, food, and other factors.Occurrence of Alport syndrome is caused by mutations in Col4α5 mutant rats of WKY strain. Some number of glomeruli exhibited capillary tuft collapse at 8 weeks of age, and more glomeruli revealed abnormalities with crescent formation and focal sclerosis at the 20 weeks of age in Col4α5 mutant males. The levels of BUN and Cre in serum highly increased in 20 weeks of age, and then died at around 24 weeks of age in hemizygous mutant males. These physiological and pathological features correspond to those of previously reported Col4α3-/- with C57BL/6 mouse strain15. However, Col4α3-/- with 129X1/Sv mouse strains showed much earlier progression of disease, died at around 12 weeks of age42. Therefore, comparison of a survival ratio between different strains of mice and rats should be carefully studied them, and further studies are required to draw a definite conclusion. In further study, other rat strains of Col4α5 mutant using the rGONAD method can be expected.In the present study, we are able to produce 16. Using this Alport model rat, there are certainly advantages compared with those of mouse and dog. Rat is cheaper than dog to breed and maintain, and much reliable than dog to design genetic study because of much number of pups and much shorter gestational age. On the other hand, rat is more readily than mouse to measure the proteinuria level. Then, rat provides enough glomeruli, blood, and urine to proceed experiments that require significant amounts of protein, facilitating proteomic approaches to understanding glomerular disease mechanisms compared with mouse. Therefore, we strongly suggest that Alport syndrome model rat with the rGONAD method can be useful in variety of experimentation for underlying the mechanism of renal diseases.In the past decades, there were several Alport syndrome models such as mouse and dog43. In some situation, rat is considered as a more relevant model among mammals. For example, physiology of rat is greatly well documented, because its larger body size affords the opportunity for serial blood draw studies. In particular, blood pressure measurement by telemetry is easier to perform and more reliable in rat compared to smaller mouse45. Moreover, there are several rat strains in order to perform specific studies, i,e, SHR for hypertension46. Although genome engineering experiments of rat have yet not been extensively proceeded, it is readily applicable to produce the gene-editing mutant rats in Col4α5 gene of the strains by the rGONAD method. The model rat might provide new insight into the study of the relationship between renal disease such as Alport syndrome and other blood pressure related diseases.The laboratory rat has long been recognized as a preferred experimental animal in wide areas of biomedical scienceCol4α5 mutant rat is a reliable candidate for Alport syndrome model animal for underlying the mechanism of renal diseases and further identifying potential therapeutic targets for human renal diseases.In conclusion, we have described creation of a novel rat model of X-linked Alport syndrome. This rat model should be available for the study of progressive renal failure having basement membrane abnormality. Thus, WKY/NCrl rats were obtained from Charles River. The rats were kept with a 12:12-h light: dark cycle. They were given free access of drinking water and food. All animals were handled in strict accordance with good animal practice as defined by the relevant national and/or local animal welfare bodies. All animal works were approved by the Shigei Medical Research Institute Animal Care Committee , and were performed in accordance with relevant guidelines and regulations. The manuscript was prepared according to the ARRIVE guidelines.30. Briefly, gene targeting strategy is designed to integrate tandem STOP codons into 27 bases after the first ATG in the rat Col4α5 gene   was used in accordance with the manufacturer’s protocol. Approximately 2–2.5 µl of electroporation solution was injected into the oviductal lumen from up-stream of ampulla using a micropipette. The electroporation was performed using a NEPA21 .47. Rat genomic DNA was isolated from ear-piece or tail. Genotyping was performed by PCR with the following primers , periodic acid Schiff (PAS), periodic acid methenamine silver (PAM) and Masson's trichrome (MT) by standard methods.31. In brief, renal paraffin sections of 4 µm thickness were stained with PAM. The sections on the slides were directly observed without a cover clip, with LVSEM  at acceleration voltage of 15 kV with 30 Pa.Under LVSEM, 3 dimensional ultrastructure of the GBM of Alport syndrome, the kidney was examined as described previously48. Ultrathin epoxy resin sections were prepared and observed using an H-7600 electron microscope (Hitachi Co. Ltd.).TEM was performed as described previouslyHis-tagged (for production of antibody) and MBP-tagged (for immunoblotting) NC1 domains of type IV collagen α1-6 (V) were expressed in BL21-CodonPlus-RP  transformed with pET-28a (Invitrogen) and pMAL , respectively. Each His or MBP fusion protein was purified through affinity chromatography with TALON metal affinity resin  or with amylose resin (New England Biolabs), respectively.50. Briefly, the antigen emulsion was injected to Col4a5 deficient males. The treated rats were sacrificed 21 days after the injection, and the lymphocytes were fused with SP2/0-Ag14 myeloma cells. After the cell fusion, culture supernatants were screened to confirm positive clones by solid-phase enzyme-linked immunosorbent assay (ELISA).We produced rat monoclonal rCOL4A6, as described previously3; Tubulin (T9026), α-SMA (1A4) ; phospho-Smad3 (ab52903), Fibronectin (ab6328), LDL receptor (ab52818), MMP12 (ab52897) ; Smad3 ; Nephrin ; Nestin ; Laminin β2 (MAB2066), OPN (AF808) ; CTGF (sc-373936), MMP3/10 (sc-374029) ; and MBP (New England Biolab). Primary antibodies were detected using species-specific secondary antibodies conjugated to either Alexa Fluor 488 or 555 (Molecular probes).The following primary antibodies were used: rat monoclonal type IV collagen α1(IV) (H11), α2 (IV) (H22), α3 (IV) (H31), α4 (IV) (H43), α5(IV) (H52) (In our institute)2) with protease inhibitor cocktail . Protein concentrations for tissue extracts were determined by Coomassie Brilliant Blue staining by SDS-PAGE gels. The lysates were loaded, transferred, and subjected to Western blotting with specific antibodies as described previously51.Rat kidneys and blood plasma were homogenized directly in SDS-PAGE sample buffer. For collagenase-solubilized renal basement membranes, rat kidneys were homogenized and incubated at 37 °C for 4 h with 1 mg kidney lysates and 200 µg (1000 U) of collagenase  in digestion buffer  donkey serum for 30 min at room temperature. Sections were then incubated with primary antibodies overnight at 4 °C followed by PBS wash and incubated with appropriate secondary antibodies for 1 h at room temperature. DNA was also stained with 1 µg/ml DAPI. Fluorescence images were obtained by confocal microscopy .Immunofluorescence analyses were examined, as described previouslySupplementary Information.


Arabidopsis thaliana FAHD-containing proteins and human FAHD1 (hFAHD1) implicated in mitochondrial dysfunction-associated senescence. Transcripts of the closest hFAHD1 orthologue in Arabidopsis (AtFAHD1a) peak during seed maturation drying, which influences seed longevity and dormancy. Here, a homology study was conducted to assess if AtFAHD1a contributes to seed longevity and vigour. We found that an A. thaliana T-DNA insertional line (Atfahd1a-1) had extended seed longevity and shallower thermo-dormancy. Compared to the wild type, metabolite profiling of dry Atfahd1a-1 seeds showed that the concentrations of several amino acids, some reducing monosaccharides, and δ-tocopherol dropped, whereas the concentrations of dehydroascorbate, its catabolic intermediate threonic acid, and ascorbate accumulated. Furthermore, the redox state of the glutathione disulphide/glutathione couple shifted towards a more reducing state in dry mature Atfahd1a-1 seeds, suggesting that AtFAHD1a affects antioxidant redox poise during seed development. In summary, AtFAHD1a appears to be involved in seed redox regulation and to affect seed quality traits such as seed thermo-dormancy and longevity.Fumarylacetoacetate hydrolase (FAH) proteins form a superfamily found in Archaea, Bacteria, and Eukaryota. However, few fumarylacetoacetate hydrolase domain (FAHD)-containing proteins have been studied in Metazoa and their role in plants remains elusive. Sequence alignments revealed high homology between two  Caenorhabditis elegans, knocking down the hFAHD1 orthologue impairs mitochondrial function, locomotion, egg-laying behaviour, and neuronal transmission , an, anAtFAHAtfahd1a-1 seeds revealed a shallower thermo-dormant phenotype than WT seeds, whereby significantly more non-stratified Atfahd1a-1 seeds germinated at 25 °C after harvest. As typical of weakly dormant winter annual species, dormancy was less expressed in both genotypes at lower germination temperatures , leading to >95% TG. Nonetheless, Atfahd1a-1 seeds still germinated faster than WT seeds at all tested temperatures  compared to Arabidopsis Col-0 accessions [2O g−1 DW, respectively) would possess a fluid-rubbery cytoplasm [2O g−1 DW) were likely in the same physical state, whereby the cytoplasm retained similar viscosity  and is accompanied by extensive metabolic rearrangements ,83. Therno acids . Mobilisturation . Particuturation ,55,83. Fturation ,86). Atf-alanine , which cn the WT , the lowc stress . As the Atfahd1-1a seeds. Firstly, reducing sugars are implicated in non-enzymatic reactions with amino acids, forming Amadori and Maillard products , which negatively correlate with longevity [Atfahd1a-1 seeds  and δ-tocopherol , which aongevity ,88,89. Songevity ,91. In Aongevity . Compare-1 seeds . Therefois study ,48,64, iAtFAHD1a resulted in dry seeds displaying altered contents of some LMW metabolites, which are implicated in the remodelling of seed metabolome during seed late maturation and desiccation.In summary, knocking out Atfahd1a-1 seeds compared to the WT   , which a values) , under wat seeds . In summary, AtFAHD1a appeared to participate in seed redox regulation during seed late maturation and desiccation.Arabidopsis thaliana L.  of the ecotype Columbia (Col-0) were compared with an insertion line of the SALK collection in the Col-0 background . Five seeds per pot (n = 10–15) were cold stratified for 4 d at 4 °C in wet commercial soil mix , before transfer to controlled growth conditions  for about 13 weeks, required for the production of fully mature seeds. At the rosette stage, plants were sheltered with Aratubes  to prevent cross-pollination between the two parental genotypes  for the production of F1 seeds. Regular watering was stopped when first siliques senesced. F1 seeds produced by individual parental plants were harvested at full maturity before equilibration over a non-saturated LiCl solution (36–38% RH) for 6 d at room temperature (RT). For long-term storage, desiccated F1 seeds were hermetically sealed in a plastic box over silica gel and preserved at −20 °C until further analyses. 50 F1 seeds for each individual plant pool were germinated for three weeks in sealed Petri dishes over three layers of filter paper , and seedlings were used for DNA extraction and PCR screening of homozygous genotypes. All heterozygous F1 seeds were disregarded, and only F1 seeds obtained from F1 plants homozygous for the T-DNA insertion or the WT gene AT4G15940 were used for both germination assays and production of a F2, grown as described for the F1. At full maturity, F2 seeds were also pooled from individual plants, grown for 3 weeks at the seedling stage and further screened for the homozygosity of both WT and Atfahd1-1 lines. Only pure F2 seed lines were included in the germination assays to reveal phenotypes.WT seeds of 020157C, ), both pAT4G15940 specific primers (left primer 5′-ACATGAGGATCATATGCCCTG-3′ and right primer 5′-CTTGAGAGAGGATTTGAGCCC-3′), primers to amplify the T-DNA insertion of the mutant line (LBb1.3 5′-ATTTTGCCGATTTCGGAACCA-3′ and RP 5′-CTTGAGAGAGGATTTGAGCCC-3′), and a REDTaq® ReadyMixTM . PCR conditions included an initial step of 5 min at 95 °C and 29 cycles consisting of 30 s at 95 °C, 40 s at 57 °C, and 1 min 40 s at 72 °C, followed by a final extension step of 10 min at 72 °C. The size of the PCR products was checked by agarose gel electrophoresis [Genomic DNA was extracted from typically 50 3-week-old seedlings (approximately 2 mg DW), using the cetyltrimethyl ammonium bromide method described by Clarke . To confphoresis .2O on three layers of filter paper  in 9-cm Petri dishes sealed with Parafilm. Germination assays were conducted at 25 °C with 16 h of light at 49 ± 5 μmol photons m2 s−1 and 8 h of dark to investigate the occurrence of primary dormancy. The light intensity and photoperiod were maintained constant across all germination assays. A seed was regarded as germinated when the radicle tip protruded through the endosperm [2 seeds of both lines were sown on filter paper soaked with 4 mL of ultrapure H2O before 4 d of cold stratification at 4 °C. Thereafter, samples were dried-back under a laminar flow bench, imbibed with ABA solutions (0–5 µM range), and incubated at 25 °C. Seeds were scored at regular intervals for both testa and endosperm rupture, the latter of which is regulated by ABA [Following equilibration after harvesting, a minimum of six replicates of 100 seeds pooled from individual plants of both lines and generations were imbibed with 4 mL of ultrapure Hndosperm , and TG ndosperm . When mendosperm . Furtherd by ABA ,113.2O. Thereafter, seeds were incubated in the dark at 40 °C and the stability of temperature and moisture conditions over time were monitored with data loggers . Seeds of both generations were tested at 59.4 ± 1.1% RH and only seeds of the F1 generation were subjected to other ageing regimes. To assess the effect of ageing under ambient temperatures on viability, seeds of the F1 generation were equilibrated and stored at indoor ambient temperatures  and 68.2 ± 2.0% RH. At intervals during the CD treatments, seeds (n = 6 replicates of 100 seeds for both lines) were incubated at 20 °C to assess their viability by scoring TG. Finally, after CD at 59.4 ± 1.1% RH for 11 weeks, the TG of F2 generation seeds was also assessed under the following conditions: (1) at 20 °C, (2) at 20 °C after 4 d of cold stratification at 4 °C, (3) at 20 °C after imbibition with 1 µM GA3, and (4) at 15 °C. Seed WCs during CD, AA, and ageing under ambient temperatures were estimated using water sorption isotherms as determined by Hay et al. [To characterise seed longevity, seeds were equilibrated at RT in hermetically sealed plastic boxes over non-saturated LiCl solutions at 59.4 ± 1.1% and 74.5 ± 0.5% RH, measured with a hygrometer  or at 100% RH using ultrapure Hy et al. . 1 (n = 4 replicates of ca. 14 mg seed fresh weight (FW) pooled from individual plants of both lines) were frozen in liquid nitrogen and lyophilised for 5 d before storage over silica gel at −80 °C in a hermetically sealed plastic box until analysis. Right before extraction, samples cooled to −80 °C were ground with two 2-mm diameter agate beads using a bead mill  set at 30 Hz for 2 min. Then, 13.39 ± 0.89 mg of finely ground seed powder were resuspended in 1 mL of pre-cooled (−20 °C) water:acetonitrile:isopropanol , containing 21.26 µM 13C6-sorbitol and 25 µM 13C6, 15N-valine  as internal standards, and LMW metabolites were extracted for 10 min at 4 °C with continuous shaking at 1000 rpm . Insoluble material was removed by centrifugation at 20,000× g for 5 min at 4 °C. Then, a 25-µL aliquot of the supernatant was collected and dried for 3 h in a vacuum centrifuge . Blanks and a standard mix were processed following the same steps as the samples and used as quality controls. Derivatisation and gas chromatography coupled to mass spectrometry (GC-MS)-based metabolite profiling were conducted according to Fiehn et al. [® MHR 13, GML, Innsbruck, Austria). Then, 90 µL of N-methyl-N-trimethylsilyl-trifluoroacetamide  was added, and the derivatisation reaction continued for 30 min at 37 °C. After cooling at RT, 1 µL of each sample was injected in splitless mode into a Trace 1300 gas chromatograph coupled to a TSQ8000 triple quadrupole mass spectrometer  and equipped with a 30-m Rxi-5SilMS column with 0.25 µm cross bond 1,4-bis(dimethylsiloxy)phenylene dimethyl polysiloxane film and a 10-m integra-guard pre-column . The analysis and data processing followed the same workflow as previously reported [TM software  for quantification. Relative values of LMW metabolite contents were obtained after normalising the peak areas of each metabolite to that of the internal 13C6-sorbitol standard and to the DW of individual samples.Dry seeds of the Fn et al. ,116, witn et al. . Vacuum-reported , using treported ) to firs1  were frozen in liquid nitrogen, lyophilised, and ground as described for GC-MS analyses. gsh, cys, γ-glu-cys, cys-gly, and their corresponding disulphides were extracted on ice from 11.74 ± 0.67 mg of lyophilised and finely ground seed powder with 1 mL of 0.1 M HCl. Approximately 12 mg of polyvinylpolypirrolidone were also included before resuspension at 30 Hz for 30 s with a bead mill . After a first centrifugation step , a 700-µL aliquot of the supernatant was promptly transferred to a new Eppendorf tube and further centrifuged . The assay is based on fluorescence labelling of thiol groups by monobromobimane  to block free thiol groups and quantify disulphide concentrations only. Excess NEM and NEM-bound-thiols were removed by washing five times with toluene, and the remaining disulphides were reduced with DTT before labelling with mBBr. LMW thiols were separated by reversed-phase HPLC using a HPLC 1100 system  with a ChromBudget 120-5-C18 column  including a NUCLEODUR C18 Pyramid guard column  and detected by fluorescence . The concentrations of LMW thiols and their corresponding disulphides were calculated using calibration curves of individual LMW thiol standards and subtracting the amounts of disulphides  from the amounts of thiols plus disulphides, as described earlier [Dry seeds of the Fe , occurr earlier .GSSG/2GSH was calculated according to the Nernst equation (Equation (1)). Seed WC was determined by measuring sample weights before and after seed lyophilisation, expressed as g H2O g−1 DW, and used to estimate the molar concentrations of GSH and GSSG according to Equation (1):R is the gas constant (8.314 J K−1 mol−1); T, temperature in K; n, number of transferred electrons (2 GSH → GSSG + 2 H+ + 2 e−); F, Faraday constant (9.649 × 104 C mol−1); E0’, standard half-cell reduction potential at cellular pH of 7.3 .2 (n = 4 replicates of ca. 10 mg of both lines) were characterised for OCRs, using a FluoRespirometer Oxygraph-2k . Before each measurement via Clark-type electrodes , chambers were calibrated to 260.82–262.23 µM O2 and corrected for baseline signal with the DatLab software . Dry seeds (9.98 ± 0.22 mg) were directly imbibed in the electrode chambers containing 2 mL of ultrapure H2O. Temperature was held constant at 21 °C, while stirring the seeds at 750 rpm in sealed electrode chambers. Data were acquired every 2 s with a polarisation voltage of 800 mV. Seed OCRs, indicative of mitochondrial respiration, were calculated during 15 min intervals in the plateau phase . Thereafter, 25 mM of KCN was added to block respiration by inhibiting complex IV. Finally, 25 µM of n-octyl-gallate, an inhibitor of the mitochondrial alternative oxidase, was injected to assess O2 consumption by this KCN-insensitive bypass of the mitochondrial ETC [Dry seeds of the Frial ETC ,120. SeeMultiple sequence alignments were conducted using Clustal Omega .post-hoc multiple comparisons of means, or Student’s t-test for comparing variables between the WT and mutant lines. All data were analyses using the software package SPSS Statistics 25 . In each dataset, values with a Cook’s distance larger than 4/n  were disregarded. The assumptions of normal distribution and homoscedasticity of variances were assessed via the Shapiro-Wilk and Levene’s tests, respectively. Whenever the assumption of normality was not fulfilled, data underwent non-parametric analyses , followed by Bonferroni correction for multiple tests. For data showing heteroscedastic variances, simple bias-corrected accelerated bootstrap analyses were run with a sample size of 105 and two different seeds , using the Mersenne Twister random number generator. The 95% confidence intervals of the bootstrap analyses’ outputs were sensitive to the seed size at the first decimal digit. Probit regression was run to interpolate  or extrapolate  the P50 values from survival curves [® 2017 .Data were tested for significance at α = 0.05 by either one-way ANOVA analyses followed by Tukey’s Honest Significant Difference (HSD) test for l curves . P50 valIn conclusion, our data support that the lack of AtFAHD1a activity during seed development can have downstream consequences on seed quality traits. Therefore, we propose AtFAHD1a as a novel candidate player in seed metabolism, which influences cellular redox poise, seed thermo-dormancy, and longevity.Atfahd1a-1 seed phenotype. The metabolic profile of dry seeds revealed that AtFAHD1a knockout influenced metabolic rearrangements typical of the late seed maturation and desiccation, in line with highest expression levels of AtFAHD1a during these stages of plant life cycle. Nonetheless, further research is required to reconcile influences on primary metabolism and the Atfahd1a-1 seed phenotype. Considering the low constitutive expression of ATFAHD1b, an ATFAHD1a paralogue sharing high sequence homology also with hFAHD1, future studies to elucidate the functional role of AtFAHD1-containg proteins may benefit from a more extensive seed phenotypic characterisation, including ATFAHD1b knockout lines and AtFAHD1a AtFAHD1b double mutants. Additionally, a full validation of the biochemical profiles of Atfahd1a-1 and WT seeds would also require accurate determination of AtFAHD1a and AtFAHD1b enzymatic activities, which may also contribute to ascertain the biochemical function of these FAHD-containing proteins in A. thaliana and generally in plant metabolism. This study focused on the characterisation of


Mycobacterium tuberculosis complex (MTBC) in treated and untreated wastewater. Wastewater samples were taken from three wastewater treatment plants (WWTPs) in the city of Durban, South Africa, known for a high burden of TB/MDR-TB due to HIV infections. All untreated wastewater samples contained total mycobacteria and MTBC at varying percentages per WWTP studied. Other members of the MTBC related to tuberculosis infection in animals, M. bovis and M. caprae were also detected. The highest median concentration detected in untreated wastewater was up to 4.9 (±0.2) Log10 copies/ml for total mycobacteria, 4.0 (±0.85) Log10 copies/ml for MTBC, 3.9 (±0.54) Log10 copies/ml for M. tuberculosis, 2.7 (±0.42) Log10 copies/ml for M. africanum, 4.0 (±0.29) Log10 copies/ml for M. bovis and 4.5 (±0.52) Log10 copies/ml for M. caprae. Lower concentrations were detected in the treated wastewater, with a statistically significant difference  in concentrations observed. The log reduction achieved for these bacteria in the respective WWTPs was not statistically different, indicating that the treatment configuration did not have an impact on their removal. The detection of M. africanum in wastewater from South Africa shows that it is possible that some of the TB infections in the community could be caused by this mycobacterium. This study, therefore, highlights the potential of wastewater-based epidemiology for monitoring tuberculosis infections.The surveillance of tuberculosis infections has largely depended on clinical diagnostics and hospitalization data. The advancement in molecular methods creates an opportunity for the adoption of alternative surveillance systems, such as wastewater-based epidemiology. This study presents the use of conventional and advanced polymerase chain reaction techniques  to determine the occurrence and concentration of total mycobacteria and members of the  Mycobacterium tuberculosis complex; Wastewater; Droplet digital PCR.Tuberculosis; Mycobacteria;  Most TBcterium) , Mycobac Africa)  and Mycof Africa  and repoM. tuberculosis from the environment, such as hospital sewage  people worldwide were infected with tuberculosis, with 1.5 million deaths per year . In mostl sewage , househol sewage  and farml sewage . Therefol sewage . This mel sewage . This apl sewage  WBE studl sewage .M. tuberculosis can be cultured from soil and other materials, but due to bacterial overgrowth and the presence of "differentially culturable" (or "viable but non-culturable") organisms, sensitivity may be limited  wastewater in KwaZulu Natal (KZN), South Africa.22.1Three wastewater treatment plants (WWTPs) in the city of Durban, South Africa, were sampled for municipal wastewater: WWTP A, WWTP B, and WWTP C, on four different occasions. The WWTPs were chosen based on whether or not they served a population of at least 10,000 people and whether or not they received hospital sewage. The treatment configurations and capacities of these WWTPs were also different. For example, WWTP A does not have an activated sludge treatment or secondary clarification process, whereas WWTP B and C both do. There are other differences in the treatment processes, as shown in 2.2Each WWTP's influent (raw/untreated wastewater) and effluent (treated wastewater) received a 1-L composite sample. Composite samples made up of many subsamples were taken, for example, one small sample was taken, followed by a 30-second interval, and then the next sample was taken, and so on, until the required sample volume (1 L) was reached. The samples were transported to the lab in an ice-filled cooler box, kept at 4 °C, and analyzed within 48 h. Per WWTP, two samples were taken (influent and effluent (post-chlorination)). Samples were homogenized before analysis, and 50 mL subsamples were taken and centrifuged at 3000 rpm for 20 min, with the supernatant discarded and the pellet used for DNA extraction. The DNA was extracted using a DNeasy Powersoil DNA extraction kit (QIAGEN) according to the manufacturer's instructions, with no changes. IMPLEN NanoPhotometer NP80 – All-in-One Spectrophotometer was used to determine the quantity and quality of the extracted DNA. All of the analyses were carried out in triplicate.2.3M. tuberculosis complex, M. tuberculosis, M. africanum, M. bovis and M. caprae. In this study, regions of differences (RDs) in these various organisms were targeted based on their uniqueness. However, it must be noted that some of these regions of differences are shared among the species within the M. tuberculosis complex and difference studies report them differently. For instance, the total mycobacteria were targeted using the 16s rRNA gene  to the following copies/μl: 59.2, 18.6, 13.75 and 7.06 respectively for conventional PCR to determine the lowest concentration of M. tuberculosis detectable.The limit of detection (LOD) of the PCR protocol was determined using positive control DNA of M. tuberculosis. The PCR mixture for all targeted organisms contained 12.5 ul of OneTaq 2X Master Mix with Standard Buffer (New England BioLabs inc), 2 l of primer mix , 1 l (60 ng/l) of DNA template, and 9.5 l of molecular grade water in a final volume of 25 l in a reaction tube. The VeritiTM 96-Well Thermal Cycler was used for PCR amplification. Optimised thermocycling conditions were initial denaturation step at 95 °C for 10 min and followed by 30 cycles of 96 °C for 45 s. The annealing temperatures varied for each primer (organisms), total mycobacterial species (16s rRNA gene- 500bp) at 61.5 °C for 45 s, M. tuberculosis complex (Rv0577-700bp) at 54 °C for 60 s, M. tuberculosis (RD9 present- 369 bp) at 59 °C for 60s, M. africanum (RD8 present- 150 bp) at 68 °C for 60 s, M. bovis (RD1 present-264bp) at 57 °C for 60s and M. caprae (RD4 present) at 58 °C for 60s and extension at 72 °C for 40 s. The final extension step was performed at 72 °C for 10 min.The lowest detectable concentration of After amplification, 3 μl of ethidium bromide  was added on 2% agarose gel in of 1× Tris-borate-EDTA (TBE) buffer and analysed via agarose gel electrophoresis. The assay products were electrophoresed for 45–60 min at 70 V in 1× TBE buffer, and the gels were visualized using a Bio-Rad Gel Doc™ XR.2.4The optimized conventional PCR protocol was used to determine the presence of the selected organisms or group of organisms in wastewater from Durban, South Africa. Wastewater samples were collected and processed using the methodology described above Section .2.5M. tuberculosis complex mentioned in section M. tuberculosis using a measured DNA template (1.5 ng/μl of M. tuberculosis H37Rv strain). The DNA template was serially diluted from 10−1 to 10−9.The Rv0577 primer for The concentration of these organisms was determined using the droplet digital PCR (ddPCR). The same set of primers presented in M. tuberculosis complex (Rv0577-700bp) at 54 °C for 60 s, M. tuberculosis (RD9 present- 369 bp) at 59 °C for 60s, M. africanum (RD8 present- 150 bp) at 68 °C for 60 s, M. bovis (RD1 present-264bp) at 57 °C for 60s and M. caprae (RD4 present) at 58 °C for 60s and final incubation step was performed at 98 °C for 10 min (ramp rate 2.2 °C/s). These conditions were applied to the wastewater samples. After thermal cycling, the ddPCR plates were read using the QX200 droplet reader (Bio-Rad). Droplet counts and amplitudes were analysed with QuantaSoft™ analysis Pro software (Bio-Rad).Droplets were generated using the automated droplet generator and the following amplification protocol was followed: Optimised thermocycling condition included an initial denaturation step at 95 °C for 10 min and followed by 30 cycles of 96 °C for 45 s. The annealing temperatures varied for each primer (organisms), total mycobacterial species (16s rRNA gene- 500bp) at 61.5 °C for 45 s, M. tuberculosis (H37Rv strain) DNA was determined to have an average of 9226 (±642.1) copies/mL using this protocol/assay. The LOD after ten-fold serial dilutions was determined to be 3.0 (±0.06) gc/ml  with an average of 18,892 droplets generated per well.The standard/reference 2.6Descriptive statistics was calculated using Microsoft Excel, and a test of normality was conducted using the Akaike Information Criterion (AIC) score, which was calculated using @Risk . The Kruskal-Wallis tests, followed by Dunn's Multiple Comparison tests, were used to compare the concentrations of different tuberculosis-causing mycobacteria based on the normality tests. The Mann-Whitney tests were used to compare the concentrations of mycobacteria in untreated and treated wastewater. All statistical tests had a 95% confidence interval, and a p-value of less than 0.05 was considered statistically significant. GraphPad Prism was used for all statistical analyses .33.1M. tuberculosis complex primer. The detected mycobacterial organisms varied between the three WWTPs. Total mycobacteria was detected in all (treated and untreated) wastewater samples analysed (M. tuberculosis complex (MTBC) was present in all (100%) untreated and majority (75%) of treated wastewater samples from all the three WWTPs  Log10 copies/ml as compared to both WWTP B (3.8 (±0.33) Log10 copies/ml) and WWTP C (3.3 (±0.52) Log10 copies/ml).Comparing the concentration of the six organisms or group of organisms measured in the untreated wastewater across the three WWTPs showed differences in their concentrations. Between the WWTPs, a statistically significant difference in concentrations of each organism was observed. The highest median concentration for total mycobacteria in the untreated wastewater was recorded in WWTP A (4.9 (±0.2) Log10 copies/ml), with the lowest median concentration of 4.8 (±0.06) and 4.8 (±0.47) Log10 copies/ml in WWTP B and WWTP C, respectively. The M. tuberculosis  were also recorded in low concentrations. However, comparing with M. bovis and M. caprae, the concentrations of M. africanum in the untreated wastewater samples were significantly lower .WWTP A and WWTP B influent had similar median concentrations of rculosis  and loweM. tuberculosis, M. africanum, M. bovis and M. caprae concentrations in the treated and untreated wastewater in all the WWTPs was not statistically significant . Despite the reductions observed in all the WWTPs, concentrations up to 4log10 for these mycobacteria are released into receiving water environments  in all the WWTPs, except WWTP C. However, the difference in MTBC, ronments .3.3M. tuberculosis complex members of 0.71 (±0.65). Despite these differences in log reduction as can be seen in The observed log reduction in each WWTP as presented in Figures M. tuberculosis was observed in WWTP B (0.36 (±0.41)) and the lowest at WWTP A (0.21 (±0.29)). In respect of M. africanum, the highest reduction was recorded at WWTP C (0.22 (±0.28)) and the lowest at WWTP A  and M. africanum West African 2 (MAF2)  . M. afrit region . Therefoh Africa , which ifricanum . The det10 concentrations per ml of wastewater. Comparatively, the concentrations recorded in our study for total mycobacteria or MTBC were higher than the results published by 10 copies/mL in untreated wastewater were reported. However, it is worth mentioning that The concentration of the mycobacteria analyzed varied both by WWTP and by organism. However, it was observed that total mycobacteria and MTBC concentrations were largely within the 4-logM. tuberculosis and M. bovis could survive for hours to days in the amoebal trophozoites (M. tuberculosis and M. bovis organisms were engulfed by Acanthamoeba polyphaga trophozoites was consistent with previous findings made when M. tuberculosis organisms were co-cultured with the free-living amoeba Dictyostelium discodium (The high concentration of the mycobacteria in the treated wastewater (up to 4 log10) could be due to resistant nature of these organisms to environmental conditions and predators. For example, tuberculosis-causing bacteria have been reported to be amoeba-resistant which may enhance their survival in the environment, especially wastewater . M. tubehozoites . The finiscodium . Additioiscodium . FurtherM. tuberculosis. These WWTPs have different treatment processes or configurations, for instance WWTP A has four processes i.e primary settling, biofilters, sludge digestion and tertiary treatment .This work was supported by the Data included in article/supplementary material/referenced in article.The authors declare no conflict of interest.No additional information is available for this paper.


Furthermore, 2A12 and 8H4 recognized distinct and non-overlapping quaternary epitopes and bound specifically with HPV18. Humanized 2A12 (Hu2A12) retained comparable neutralizing activity against HPV18 infection in various acidic pH settings and in hydrogel formulation with IC50 values of 0.04 to 0.77 ng/ml, indicating that Hu2A12 will be a promising candidate for clinical development as a topical vaginal biopharmaceutical agent against HPV18 infection.Cervical cancer caused by human papillomavirus (HPV) infections is the fourth most common cancer in women worldwide. Current prophylactic HPV vaccines have achieved promising success in preventing HPV infection. However, still 570,000 new cases were reported in 2018. The current primary treatment for the patient with cervical cancer is either surgery or chemoradiotherapy. Cervical cancer still lacks standard medical therapy. HPV18 induced cervical cancer has the worst prognosis and high mortality compared to other HPV infections. The development of HPV18 related with cervical malignancy requires the persistent infection of cervical–vaginal epithelium by HPV18 subtype, which can take years to transform the epithelium. This period of repeated infection provides a window for therapeutic intervention. Neutralizing antibodies formulated as topical agents that inhibit HPV18 infection should reduce the chance of cervical malignancy. We previously demonstrated that potent neutralizing anti-sera against HPV18 infection were induced by HPV18 viral like particle (VLP) generated in mammalian cells. We, therefore, isolated two potent neutralizing antibodies, 2A12 and 8H4, from over 3,810 hybridomas prepared from mice immunized with HPV18 VLP. 2A12 and 8H4 exhibited excellent potency, with 50% virus-inhibitory concentrations (IC Two neutralizing antibodies against HPV18 with the highest potency were isolated from immunized mice.50 value of 0.04 ng/ml.The humanized antibody, Hu2A12, exhibits ultrahigh potency against HPV18 infection with ICHu2A12 retains comparable neutralizing activity in various acidic pH settings and in hydrogel formulation.Hu2A12 will be a promising candidate as a topical vaginal biopharmaceutical agent against HPV18 infection.Cervical cancer is the fourth most common tumor diagnosed in women worldwide . PersistCurrent prophylactic HPV vaccines have achieved remarkable success in preventing HPV infection , 5. HowePersistent HPV infection in the basal layer of the cervical epithelium is the main risk factor in the development of the premalignant conditions of cervical intraepithelial neoplasia or adenocarcinoma in situ. Without treatment, the transition from dysplasia to invasive carcinoma may take years to decades to develop in most women.Current research on topical therapies for the treatment of HPV or CIN have promising results, signified by the randomized trials of immune-modulating (imiquimod), anti-proliferative (5-fluorouracil), and anti-viral (cidofovir) therapies –10. HoweThe use of neutralizing antibodies with high potency and low toxicity have been widely applied to treat viral infections caused by a respiratory syncytial virus, cytomegalovirus, human immunodeficiency virus, Ebola virus and influenza virus , 14. Thein situ compared to lower grades of diagnosis  retains comparable neutralizing activity against HPV18 infection in various acidic pH conditions and hydrogel with the IC50 of 0.04 to 0.7 ng/ml, indicating that Hu2A12 will be a promising candidate for clinical development as topical vaginal biopharmaceutical agents in hydrogel against HPV18 infection.After HPV16, HPV18 infection is the second most carcinogenic HPV genotype in a large percentage (approximately 10%), and highly enriched in adeno/adenosquamous and adenocarcinoma iagnosis , 15, 16.iagnosis , 18. Twoiagnosis . To our iagnosis . 2A12 anHPV18 VLP and pseudovirus were prepared using the plasmid of p18sheLL  and the co-transfection of p18shell and pGMCMV-luc (Yeasen Biotech) as a reporter gene, respectively, as we previously described , with soFive 8-week-old female Balb/c mice (M1~M5) were immunized by three subcutaneous injections, at two weeks interval, of 25–50 μg HPV18 VLP emulsified with Freund’s adjuvant . Sera were collected one week after the third immunization and were used to further analysis. An intraperitoneal injection of HPV18 VLP without adjuvant was performed as the booster immunization before hybridoma.To measure the serum titer or antibody against HPV18 VLP, ELISA plates  were coated with 0.5 μg/ml purified HPV18 VLP at 4°C overnight, then blocked in 2% bovine serum albumin-PBST. Sera were diluted in 2% bovine serum albumin-PBST. Diluted sera or hybridoma culture supernatant was added and then incubated at 37°C for 1.5 h. After a wash with PBST, anti-mouse IgG HRP conjugated antibodies (Jackson ImmunoResearch115-035-003) were added at 37°C for 60 min. For visualization, 100 μl 3,3′,5,5′-Tetramethylbenzidine  substrate was added and incubated at room temperature for 10 min, and stopped with 50 μl 1 M HCl per well. Optical densities were determined at 450 nm using Infinite 200 (Tecan).Mice were sacrificed 3 days after booster immunization. Splenocytes were isolated, fused with Sp2/0 cells (ATCC) in a 1:2 ratio using Electro Cell Manipulator  as described , 22. Fus50 value was calculated based on the previously published standard algorithm  and boiled for 5 min. For reduced HPV18 VLP, 5x SDS-PAGE reduce sample buffer  was added. The proteins were separated by electrophoresis on 10% SDS-PAGE gels, and the gels were then transferred onto a 0.45-μm PVDF membrane. . The membrane was soaked in blocking buffer (2% BSA in PBST) at room temperature for 1 h, and then incubated with hybridoma supernatant or anti-sera at room temperature for 2 h, followed by three times of washing. A secondary antibody of anti-mouse IgG with an IRDye 800CW  was used and the immunoblots were visualized using an Odyssey Infrared Imaging System (LI-COR).Mouse antibody isotypes were determined using mouse monoclonal antibody subtype identification Kit , following the manufacturer’s protocol. In brief, a sample of evaluated antibody was added in the ELISA plate. After incubation and washing, the secondary antibodies specific for IgG1, IgG2a, IgG2b, IgG3, IgA and IgM were added. The high optical density (450 nm) suggests the right antibody isotype or subtype.® 488-AffiniPure Goat Anti-Mouse IgG (H + L) . After washing, the cells were resuspended in 500 µl PBSF and analyzed using ACEA NovoCyte TM (Agilent Biosciences) Nontransfected 293TT cells were served as a negative control.293TT cells were transfected with the plasmid of p18sheLL to express VLP. Some 48 h later, cells were collected, fixed in formalin for 20 min, treated with 0.2% TritonX-100 for 10 min and then blocked in PBS with 1% FBS (PBSF) for 2 h. 2A12 and 8H4 were added and incubated for 1 h at room temperature. After washing twice with PBSF, binding antibodies were detected by an incubation at 4°C for 30 min with Alexa FluorImmunoelectron microscopy (Immune-EM) of the HPV virion was performed as described previously . For sta7 hybridoma cells were collected and washed with PBS, then lysed with Trizol  to extract total RNA according to the manufacturer’s instructions. RNA was reverse transcribed to cDNA using PrimeScript™II 1st Strand cDNA Synthesis Kit . The resulting cDNA was used as the template for amplifying heavy and light-chain variable gene using the Mouse Ig-Primer Set . The gene was then sequenced, adopting standard methods (http://imgt.cines.fr) was used to analyze the variable domain VH/VL. To confirm the sequences, the vectors of pCDNA3.4-VH-CH and pCDNA3.4-VL-CL containing the gene of VH and VL were constructed and co-transfected 293TT cells. The cell supernatant was then detected by ELISA as described above.Some 1 ∗ 10 methods . The IntThe mouse IgG humanization was achieved by complementarity determining regions (CDR) grafting , 27. The2 fragment antibody produced in goat) was coated. After blocking, series gradient diluted sample and mouse IgG standards (0–100 ng/ml) were added as primary antibodies, while anti-mouse IgG (Fc specific)–peroxidase antibody  was used as a secondary antibody. The linear regression curve of the mouse IgG standards was drawn, and the OD450 value was brought into the equation to calculate the mouse IgG concentration of each sample.The mouse IgG in serum or in hybridoma supernatant was quantified using double antibody sandwich ELISA. Briefly, the anti-mouse IgG The affinity of HPV18 specific antibodies was determined using the Octet RED96 instrument (Sartorius). Amine reactive biosensor  was used to immobilize the HPV18 VLP. AR2G biosensors were activated for 7 min with EDC (400 mM)/NHS (100 mM), and then immersed 15 min in HPV18 VLP which was diluted in pH 5.0 10 mM sodium acetate buffer. Transfer the sensor to ethanolamine for 7 min and then to 0.02% PBST for affinity assay. The kinetics assays were performed with a shaking speed of 1,000 rpm. Association with HPV18 VLP specific antibodies was measured for 4 min and dissociation in 0.02% PBST for 4 min. The affinity analysis was performed using a fast 1:1 binding model and the Data analysis software 8.0 (Sartorius).The epitope-binding assay was performed with AR2G biosensor following the manufacturer’s protocol ‘in-tandem assay’. The immobilization of antigen was performed as above described, and then associated the first antibody (20 μg/ml) for 400 s following with the baseline step with 30s immersion in 0.02% PBST. Accordingly, the sensors were immersed for 400s with a second antibody at 20 μg/ml. Graph Pad was used to illustrate the time-response course of two antibodies binding to HPV18 VLP.2O at a final concentration of 1 mg/ml. Subsequently, 70 mg Hydroxyethyl cellulose  was added to the 1 ml above antibody solution containing Hu2A12, which shook for 1 h at room temperature, accordingly, 7% (wt/vol) HEC hydrogel containing antibodies was prepared.To generate a hydrogel formulation contained antibodies, Hu2A12 was diluted in sterile ddHIn vitro cumulative percentage release was calculated using the formula as below:For drug release studies, 7% HEC containing Hu2A12 labeled with FITC (hu2A12-FITC) was prepared –30. TranTo apply the antibodies under acidic conditions, we firstly detected the neutralization activity of Hu2A12 in the media of pH 4, 5, 6, and 7. The complete DMEM media was adjusted with acidic acid to pH 4, 5, 6, and 7. The neutralization assay was similar to the ‘HPV neutralization assay’ as described above with some changes as follow. Both antibodies and pseudovirus were diluted in corresponding acidic media. Four hours after adding the mixture of antibodies and pseudovirus in the seeded cells, the acidic media was replaced with conventional media to avoid the cells from acid toxicity. Some 48 h later, neutralizing activity was measured.The Hu2A12 released from vaginal hydrogel was evaluated for neutralization activity. Sodium acetate buffer (pH5.0) was applied as an antibody released buffer to mimic the acidic environment of the vagina. About 1.5 fold volume of acidic buffer was added to the antibody hydrogel, making the gel disintegrated completely in 37°C, which was maintained for 72 h. At 24 and 72 h, the released antibodies were sampled for neutralization assay.®750 SE  (named Hu2A12-750). Nine female nude mice  were divided into three groups. Three mice in Group 1 were intra-vaginally treated with 50 μg Hu2A12-750 hydrogel, while three mice in Group 2 were injected with 50μg liquid Hu2A12-750 in the same way. The remaining three mice were used as negative control. Far infrared images were observed at 15 min, 4 h, 8 h, 24 h and 48 h with a small animal imaging system (NightOWL LB 983 NC100) at Ex: 740 nm/Em:780 nm. Images were captured by the CCD camera embedded in the imaging system and analyzed using Indigo imaging software Ver. A 01.19.01.Hu2A12 was labeled with far infrared dye YFGraphs were generated by GraphPad Prism 5.01 software or OriginPro 8.5 software (Origin-Lab). One- or 2-way ANOVA was performed for group comparisons. P <0.05 was considered statistically significant with data shown as mean ± SEM or mean ± SD or median + range.6 dilution) anti-sera from five immunized mice specific for HPV18 VLP protein was achieved after the third immunization (450 nm >1.0) for HPV18 VLP binding, yielding an overall hit rate of 8.7% , National Science Foundation of China (NSFC) , Nanjing University-Ninxia University Collaborative Project (Grant# 2017BN04 to ZW), Research Foundation of JiangSu Commission Health project (Grant# ZDA2020014 to XW), and Jiangsu province “Innovative and Entrepreneurial talent” and Six Talent Peaks Project of Jiangsu Province.Author LZ was employed by the company Abrev Biotechnology Co., Ltd. Author HS was employed by Y-Clone Medical Science Co. Ltd. A patent application on 2A12 was submitted by Y-Clone Medical science Co. Ltd., under CN201911358261X.The remaining 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.


Amyotrophic lateral sclerosis (ALS) is a devasting neurodegenerative disorder for which no successful therapeutics are available. Valproic acid (VPA), a monocarboxylate derivative, is a known antiepileptic drug and a histone deacetylase inhibitor.To investigate whether monocarboxylate transporter 1 (MCT1) and sodium-coupled MCT1 (SMCT1) are altered in ALS cell and mouse models, a cellular uptake study, quantitative real time polymerase chain reaction and western blot parameters were used. Similarly, whether VPA provides a neuroprotective effect in the wild-type  and ALS mutant-type  NSC-34 motor neuron-like cell lines was determined through the cell viability assay.3H]VPA uptake was dependent on time, pH, sodium and concentration, and the uptake rate was significantly lower in the MT cell line than the WT cell line. Interestingly, two VPA transport systems were expressed, and the VPA uptake was modulated by SMCT substrates/inhibitors in both cell lines. Furthermore, MCT1 and SMCT1 expression was significantly lower in motor neurons of ALS (G93A) model mice than in those of WT mice. Notably, VPA ameliorated glutamate- and hydrogen peroxide-induced neurotoxicity in both the WT and MT ALS cell lines.[Together, the current findings demonstrate that VPA exhibits a neuroprotective effect regardless of the dysfunction of an MCT in ALS, which could help develop useful therapeutic strategies for ALS. SOD1), fused in sarcoma (FUS), TAR DNA-binding protein (TDP), and chromosome 9 open reading frame 72 (C9ORF72) Valproic acid  was purchased from American Radiolabeled Chemicals Inc. . All other chemicals and reagents were purchased from Sigma-Aldrich  and are labelled as a high-grade commercial product.[Male transgenic ALS mice of the mSOD1 (G93A) H1 high-expresser strain  were bred with females with a similar background (B6/SJLF1). Postmortem spinal cord tissue sections were used in this study. The animal study protocol has been approved by the institutional animal care and use committee of the Korea Institute of Science and Technology .WT)] and mutant-type  cell lines were cultured following previously described methods VPA was dissolved in the ECF buffer in the presence or absence of specific inhibitors or substrates. Then, the uptake experiment was carried out on the NSC-34 cell lines following the designated time and pH. The uptake rate [cell-to-medium ratio (µL/mg protein)] was calculated using the following formula , respectively [+ free buffer was created to assess the effect of ions (Na+) on the [3H]VPA transport into the NSC-34 cell lines. Similarly, to observe the effect of pH, the ECF buffer was adjusted to pH 6.0–7.4 using HCl or NaOH following a previously described experimental procedure VPA uptake for 10 s at pH 7.4. Then, the kinetic parameters were calculated and fitted to the Michaelis–Menten constant (Km) and maximum uptake rate (Vmax) through the non-linear least-squares regression analysis using Eq.  values were calculated by converting the net [3H]VPA uptake to percentage of inhibition in the presence of edaravone, ibuprofen, and salicylic acid. The obtained values were plotted using Sigma plot version 12, and were subjected to nonlinear regression .Similarly, half maximal inhibitory concentration . Then, 25 µg of protein was subjected to SDS/10% PAGE and blotted with primary antibodies  [MCT1 (SLC16A1)  and SMCT1 (SLC5A8/AIT) ]. β-Actin (ACTB) was used as a protein loading control   on the same membrane.To determine MCT1 (SLC16A1) and SMCT1 (SLC5A8/AIT) immunoreactivity changes, WT littermate (n = 5) and SOD1 (G93A) mice (n = 5) at 120–150 days of age were perfused using 4% buffered paraformaldehyde in PBS. Their spinal cords were extracted and serially sectioned at 30 µm thickness using a cryostat. Lumbar spinal cord sections  were used for immunostaining to detect MCT1 and SMCT1 levels. First, the tissue sections were incubated for 1.5 h in a blocking solution  and were further incubated with primary antibodies MCT1(SLC16A1) (1:200 dilution)  and SMCT1 (SLC5A8/AIT). (1:200 dilution) , BAX  (1:100 dilution), and BCL2  (1:100 dilution) in a blocking solution at 4 °C for 24 h. After washing thrice with PBS, the tissue slides were processed with a Vector ABC Kit . The MCT1 (SLC16A1) and SMCT1 (SLC5A8/AIT) signals were developed using the DAB chromogen . Images were then analyzed using an Olympus microscope system . The semi-quantification of immunoreactivity was analyzed using ImageJ. In particular, the intensity of MCT1 (SLC16A1) and SMCT1 (SLC5A8/AIT), BAX, and BCL2 signals was counted from five to 10 motor neurons per section. A total of five sections (section/case) were used for the semi-quantification.3H]VPA in NSC-34 cell lines, mMCT1 (SLC16A1), mSMCT1 (SLC5A8), and SMCT2 (SLC5A12) transporters and the non-targeting pool (control) siRNAs  with Lipofectamine® 2000  were selected at a concentration of 200 nM. Using the siRNAs, NSC-34 WT and MT cell lines were transfected and incubated at 37 °C. Following the transfection, [3H]VPA uptake was performed for 10 s under physiological pH. Western blot analysis was also done using both the NSC-34 WT and MT cell lines  solution (5 mg/mL) was added and the cells were stored in the dark at 37 °C until the appearance of a purple precipitate (formazan) VPA transport characteristics, a time course of uptake was performed in WT and MT ALS cell lines for 5 to 30 s at an acidic pH (6.0) at 37 °C. The VPA uptake was demonstrated to be time dependent, with the transport rate rapidly and linearly increasing up to 10 s in both WT and MT ALS cell lines  to physiological (7.4). The VPA transport system was shown to be pH sensitive. The uptake rate decreased with the pH increase from acidic to physiological in both cell lines  and the maximum transport velocity (Vmax) at two sites. At the high-affinity sites, the obtained Km1 values for WT and MT ALS cell line was 37.8 ± 3.0 and 19.1 ± 0.7 µM, respectively. The Vmax1 values for the WT and MT ALS cell lines were 0.0083 ± 0.0044 and 0.0232 ± 0.0007 nmol/mg protein/10 s, respectively. At the low-affinity sites, the Km2 and Vmax2 values were 4.59 ± 0.62 and 1.14 ± 0.14 mM and 1.86 ± 0.24 and 0.231 ± 0.015 nmol/mg protein/10 s for the WT and MT ALS cell lines, respectively , SA, 4-phenylbutyric acid (PBA), and α-cyano-4-hydroxycinnamate (CHC) at concentrations of 1 and 10 mM strongly inhibited the VPA uptake in both WT and MT ALS cell lines compared with that in the respective controls. In addition, the organic anion transporter (OAT) substrate/inhibitor para-aminohippuric acid (PAH) significantly reduced the VPA uptake. However, a tricarboxylate (citric acid) did not have a significant effect on the uptake of VPA in both WT and MT ALS cell lines  the VPA uptake by both the WT and MT ALS cell lines at concentrations of 1 and 10 mM. The OAT substrates edaravone (drug for ALS) and estrone 3-sulfate (E-3S) also significantly reduced the VPA uptake rate. Moreover, furosemide, a loop diuretic, and 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid (DIDS), an anion exchange inhibitor, caused noticeable depletion (approximately 40%) in VPA transport at a concentration of 1 mM both in WT and MT ALS cell lines  cell lines. The protein expression pattern of MCT1 and SMCT1 were depleted in the MT cell lines than in the WT (control) cell lines  ALS mice Fig. .Fig. 4PrImmunoreactivity of MCT1 and SMCT1 was mainly observed in motor neurons, and their levels in the spinal cord gray matter were lower in ALS G93A) mice than in WT littermate mice Fig. a–d, whicA mice thSMCT1 and MCT1 siRNA transfection affected the protein expression levels in the NSC-34 cell lines. After 48 h of siRNA transfection, the protein expression pattern was evaluated through Western blot analysis  and Smct2 (Slc5a12) siRNAs. Afterward, [3H]VPA uptake was performed for 10 s at the physiological pH. Both SMCTs were selected because VPA is a monocarboxylate and its uptake was shown to be sodium dependent in both WT and MT cell lines .VPA is a primary substrate in the mitochondrial fatty acid β-oxidation pathway . It has 3H]VPA uptake by motor neurons was time, pH, and sodium dependent  cells and in the biomedical model Dictyostelium was shown to be time and pH sensitive VPA transport into the WT cell lines, but SMCT1 primarily facilitated the transport into the MT ALS cell lines  of drugs such as ibuprofen, SA, and edaravone were needed to block the biological process of the [ine Fig.  SA is a ubstrate . Accordi-5 cells , 37. It 2O2-induced neurotoxicity in both the WT and MT ALS cell lines 2O2-inducdel Fig. .In conclusion, the expression of MCTs (MCT1 and SMCT1) and the function of VPA transport systems were altered in ALS cellular and animal models, indicating that MCT1 and SMCT1 could be pathological markers for ALS. SMCT1 primarily facilitated the VPA transport in the ALS cell line model. VPA showed a neuroprotective role against oxidative stress and excitotoxins in the ALS cell line model. Together, VPA could be used to treat the motor neuron disease, ALS.