With a careful adjustment of the relative doping levels of the lanthanide ions and bridging ligands, the color of the luminescence was modulated, while at the same time the photophysical characteristics of the two chromophores were retained. In addition, the photophysical properties of the parent MOF (1) and its doped counterparts with various dabpdc2−/bpdc2− and Eu3+/La3+ ratios and the photoinduced energy transfer pathways that are possible within these materials are discussed. Finally, the temperature dependence study on the emission profile of a doped analogue containing 10% dabpdc2− and 2.5% Eu3+ (7) is presented, highlighting the potential of this family of materials to behave as temperature sensors.In this study, we report the luminescence color tuning in the lanthanide metal-organic framework (LnMOF) ([La(bpdc)Cl(DMF)] ( With careful adjustment of the Eu3+ doping percentage while keeping the dabpdc2− doping level at 10%, luminescence color tuning from blue to red through purple was achieved. In addition, a temperature dependent luminescence study of material 7  shows that good temperature sensing action can be obtained in the region from 80 to 180 K with the sensitivity parameter reaching the maximum value 2.51 %K−1 at 80 K.In this contribution, we report the preparation and study of a homologous series of ligand and metal co-doped lanthanide-organic frameworks where a parent framework [La(bpdc)Cl(DMF)] (1)  is dopedEu3+ ion ,15,16; Cl(DMF)] which is isostructural to the compound of the same formula reported by Hou et al. in 2013 -4,4′-dicarboxylic acid (H2dabpdc). Indeed, we found that the presence of up to 33 mol% of H2dabpdc in the initial reaction mixture leads to crystalline products which are isostructural to 1  to 1H-NMR analysis after it was digested in a mixture of D2O/NaOH. From the ratio of the peak integrals corresponding to bpdc2− and dabpdc2− . Crystal and refinement data can be found in 9 is virtually identical to that of 1 with the difference that the bridging ligand shows significantly greater disorder and had to be refined in two positions  under air . The framework remains thermally stable up to ~500 °C and then the second mass loss step appears, corresponding to the decomposition of the framework, which is completed up to ~525 °C.The thermal stability of  air see . All ther  shows that the lowest energy absorption feature is at 375 nm and tails off rapidly after 400 nm, thereby showing virtually no absorption in the visible region (1 is doped 10 mol% with the diamino derivative dabpdc2− (2), a rather small red shift in the fluorescence peak which maximizes at ca. 463 nm was observed. Based on a comparison with our previous work on the fluorescence properties of Ca2+ and Sr2+ MOFs featuring (NH2)2bpdc2− as bridging ligand, we attribute the red shifted emission signal of 2 predominantly to the fluorescence from the dabpdc2− chromophore  (1)  can be eStarting materials and solvents were purchased from the usual commercial sources (Sigma-Aldrich and Alfa Aesar) and were used as received.Dimethyl-2,2′-dinitro--4,4′-dicarboxylate. Dimethyl-biphenyl-4,4′-dicarboxylate  was added into concentrated H2SO4 (10 mL). The mixture was stirred at room temperature for 10 min. Nitric acid  was added into concentrated H2SO4 (2 mL). This solution was added dropwise into the first mixture at room temperature over a period of 20 min. The mixture was stirred at room temperature for 4 h and then poured into ice (300 mL) to form a beige solid. The resulting solid was dissolved in dichloromethane and the aqueous phase was extracted with dichloromethane (3 × 70 mL). The combined organic layers were dried over Na2SO4 and evaporated under reduced pressure to afford a beige solid. The crude mixture was recrystallized from 2-propanol and washed with diethyl ether to give the pure product. Yield: 1.2 g . 1H NMR : δ (ppm): 8.90 , 8.37 , 7.40 , 4.02 .Dimethyl-2,2′-diamino--4,4′-dicarboxylate. Dimethyl-2,2′-dinitro--4,4′-dicarboxylate  was dissolved in 20 mL acetic acid and the solution was stirred under Ar atmosphere for 10 min. To this solution was added iron powder  and the resulting mixture was stirred at room temperature for 24 h. The suspension was filtered through celite, washed with 40 mL acetic acid and the filtrate was concentrated under reduced pressure. The solid was dissolved in ethyl acetate (80 mL) and was extracted with saturated aqueous sodium carbonate solution (2 × 80 mL) and H2O (3 × 80 mL). The combined organic layers were dried over Na2SO4 and the solution was evaporated under vacuum to yield the product as a yellow solid. Yield 900 mg . 1H NMR : δ (ppm): 7.42 , 7.21 , 7.06 , 4.97 , 3.81 .2dabpdc).2,2′-Diamino--4,4′-dicarboxylic acid  was dissolved in THF (20 mL) and 20 mL of aqueous NaOH (0.6 M) were added dropwise under vigorous stirring. The mixture was stirred overnight at room temperature. The organic solvent was removed under vacuum and the aqueous solution was acidified with acetic acid to yield a light brown solid . 1H-NMR : δ (ppm) = 12.64 , 7.40 , 7.20 , 7.40 , 4.90 .1). LaCl3·7H2O  and biphenyl-4,4′-dicarboxylic acid  were added in DMF (3 mL) and stirred until the solids were fully dissolved. The resulting solution was sealed in a screw cap 23 mL scintillation vial and placed in a preheated oven at 110 °C where it remained undisturbed for 24 h before being cooled to room temperature. Colorless needle-like crystals were isolated by filtration, washed with DMF (5 × 3 mL), and dried under vacuum overnight. Yield 32 mg (45%).Synthesis of [La(bpdc)Cl(DMF)] (1−xEux(bpdc)1−y(dabpdc)yCl(DMF)] series. LaCl3·7H2O  and biphenyl-4,4′-dicarboxylic acid  were added in DMF (3 mL) and stirred until the solids were fully dissolved. Standard solutions of EuCl3·6H2O (10−2 M) and H2dabdc (10−2 M) in DMF were prepared and calculated volumes were added in the reaction feed using a volumetric pipette, while the mixture was magnetically stirred, to achieve the desired La3+/Eu3+ and H2bpdc/H2dabpdc molar ratio. Otherwise, the procedure was identical to that for the synthesis of 1. Colorless or pale-yellow needle-like crystals were isolated by filtration, washed with DMF (5 × 3 mL) and dried under vacuum overnight. Exact percentages of Eu3+ and dabpdc2− in each doped analogue and reaction yields are shown in Synthesis of the [LaPhotoluminescence spectra. The emission spectra were measured on a Horiba fluorescence spectrometer equipped with a powder sample holder. The light source was a 450 W Xenon Arc Lamp (220–1000 nm) and the detector a red sensitive Hamamatsu R928 photomultiplier tube. All spectra were corrected for instrument response using the correction function generated after calibration of the instrument with a standard light source. Appropriate long pass filters were used to remove scattering from the sample and the monochromators. Temperature-dependent photoluminescence measurements were carried out in the 80–300 K range by placing the samples in the cold finger of a Janis VPF liquid nitrogen optical cryostat.1H-NMR. 1H-NMR spectra were recorded at room temperature on NMR Agilent 500 MHz, with the use of the solvent proton as an internal standard and on an Avance Brucker NMR spectrometer (500 MHz).α source (λ = 1.5418 Å).PXRD measurements. PXRD diffraction patterns were recorded on a Shimadzu 6000 Series X-ray diffractometer with a Cu K2 using full-matrix least-squares with SHELXL-2014/7 [9 are summarized in X-ray Crystal Structure Determination. Single crystal X-ray diffraction data were collected on a Rigaku Oxford-Diffraction Supernova diffractometer, equipped with a CCD area detector utilizing Cu Kα (λ = 1.5418 Å) radiation. A suitable crystal was mounted on a Hampton cryoloop with Paratone-N oil and transferred to a goniostat where it was cooled for data collection. Empirical absorption corrections (multiscan based on symmetry-related measurements) were applied using CrysAlis RED software . The strL-2014/7  within tL-2014/7  platformL-2014/7 , CrysAliL-2014/7 , and MERL-2014/7  for mole−1.Thermogravimetric Analysis (TGA). Thermal stability studies were performed with a Shimadzu TGA 50 thermogravimetric analyzer. Thermal analysis was conducted from 25 to 800 °C under air with a heating rate of 10 °C min


Possible contributions of Fe2+ as a ribosomal cofactor have been largely overlooked, despite the ribosome's early evolution in a high Fe2+ environment, and the continued use of Fe2+ by obligate anaerobes inhabiting high Fe2+ niches. Here, we show that (i) Fe2+ cleaves RNA by in-line cleavage, a non-oxidative mechanism that has not previously been shown experimentally for this metal, (ii) the first-order in-line rate constant with respect to divalent cations is >200 times greater with Fe2+ than with Mg2+, (iii) functional ribosomes are associated with Fe2+ after purification from cells grown under low O2 and high Fe2+ and (iv) a small fraction of Fe2+ that is associated with the ribosome is not exchangeable with surrounding divalent cations, presumably because those ions are tightly coordinated by rRNA and deeply buried in the ribosome. In total, these results expand the ancient role of iron in biochemistry and highlight a possible new mechanism of iron toxicity.Divalent metal cations are essential to the structure and function of the ribosome. Previous characterizations of the ribosome performed under standard laboratory conditions have implicated Mg Hundreds of Mg2+ ions mediate ribosomal RNA (rRNA) folding and ribosomal assembly, in some instances binding to specific sites in the universal rRNA common core by direct coordination  and small ribosomal subunit (SSU)  , stabiliit (SSU) , maintaiit (SSU)  and linkit (SSU) . Mg2+ alcleavage , an RNA cleavage .2+ in the biosphere and hydrosphere. Under pre-GOE conditions, Fe2+ would not have caused the damage to biomolecules that occurs today in the presence of O2, via Fenton chemistry  appears to be over 200 times greater for rRNA with Fe2+ than with Mg2+. The in-line mechanism of cleavage by Fe2+ was validated here by a variety of methods including reaction product characterization.Here, we demonstrate that Feetal ion ,39, the 2+ cleave RNA, they are nonetheless essential for folding and function. In parallel with our experiments comparing Fe2+ and Mg2+ in-line cleavage, we investigated whether ribosomes from Escherichia coli grown in pre-GOE conditions associate functionally with Fe2+in vivo. We grew E. coli in anoxic conditions with ample Fe2+ in the growth media. We purified ribosomes from these bacteria and have probed their interactions with metals. We identified tightly bound M2+, which survive ribosomal purification. A small fraction (∼2%) of Fe2+ ions are not exchangeable with Mg2+ in solution and are detectable after purification involving repeated washes in high [Mg2+] buffers. We use these tightly bound ions as reporters for more general M2+ association in vivo. The data are consistent with a model in which certain M2+ ions are deeply buried and highly coordinated within the ribosome (2+ ions compared to ∼1 Fe2+ ion in ribosomes from standard growth conditions. Ribosomes washed with Fe2+ contained significantly higher Fe2+ and showed more rRNA degradation than ribosomes washed with Mg2+. Our combined results show the capacity for Fe2+ to (i) associate with functional ribosomes in vivo and in vitro and (ii) mediate significant non-oxidative damage. Our results have significant implications for the evolution of rRNA and iron toxicity in disease.While metals such as Mgribosome . Our resA260 (1A260 = 40 μg rRNA ml−1).A one-tenth volume of sodium acetate  and an equal volume of 25:24:1 phenol:chloroform:isoamyl alcohol  were added to purified ribosomes. The sample was vortexed and spun at 16,200 × g in a table-top centrifuge for 5 min. The top aqueous layer was transferred to a new tube and extracted twice with a 24:1 mixture of chloroform:isoamyl alcohol (Acros Organics) using the same procedure. rRNA was then precipitated by adding two volumes of 100% ethanol, followed by incubation at −20°C for 30 min. Precipitated rRNA was pelleted by centrifuging at 16,100 × g for 15 min. The pellet was washed with 70% ethanol and suspended in 0.1 mM sodium–EDTA (pH 8.0). rRNA concentrations were quantified by E. coli ribosomes (New England Biolabs). All in-line cleavage reaction solutions were prepared and incubated in an anoxic Coy chamber . Fe2+ and Mg2+ solutions were prepared by dissolving a known mass of FeCl2·4H2O or MgCl2 salt in degassed water inside the Coy chamber. rRNA (0.5 μg μl−1) was suspended in degassed 20 mM N-(2-hydroxyethyl)piperazine-N’-2-ethanesulfonic acid , 30 mM KCl, 5% (v/v) glycerol [Invitrogen (UltraPure)], and either 25 mM of MgCl2 or 1 mM of FeCl2. Reactions were placed on a 37°C heat block and incubated for 4 days for MgCl2 and no M2+ and for 8 h for FeCl2. At each time point  4.5 μl aliquots were combined with 0.5 μl of 1 M sodium phosphate buffer pH 7.6 to precipitate the Fe2+ or Mg2+ from solution and stored at −80°C. Aliquots were defrosted on ice and combined with 2X Gel Loading Buffer II (Amicon) then loaded onto a 1% Tris/borate/EDTA agarose gel and run at 120 V for 1.25 h. The RNA in the gel was stained with GelStar™ (Lonza) and imaged with an Azure 6000 Imaging System (Azure Biosystems). Azurespot software was used as a pixel counter to create lane profiles. rRNA peaks were integrated by fitting to an Exponentially Modified Gaussian distribution using Igor Pro (v 7.08) (kobs) were found by taking the negative of the slope from the natural logarithm of the normalized peak area versus time plot. Reaction rate constants (k) were calculated by k = kobs/[M2+].Nuclease-free water (IDT) was used in all experiments involving purified or transcribed RNA. rRNA for in-line cleavage experiments was purified as above by phenol-chloroform extraction followed by ethanol precipitation of commercial (v 7.08) . Observe2+ and Mg2+ solutions were prepared by dissolving known amounts of FeSO4·7H2O or MgSO4 in degassed nuclease-free water inside the Coy chamber. To initiate the reaction, 1 mM  of Mg2+ or Fe2+ was added to 0.02 μg μl−1 a-rRNA in 20 mM HEPES–Tris (pH 7.2) in a 37°C heat block. Samples were removed at 0, 0.25, 0.5 and 1 h for added Fe2+, and at 24 h for added Mg2+. Divalent cation chelation beads (Hampton Research) were added to quench the reactions. The chelation beads were removed using spin columns. RNA cleavage products were visualized using denaturing PAGE  run at 120 V for ∼1.3 h stained with SYBR Green II.a-rRNA , which iE. coli ribosomes (New England Biolabs) was obtained by phenol-chloroform extraction and ethanol precipitation as above. A stock solution of Fe/EDTA was prepared inside the Coy chamber by dissolving a known amount of FeCl2·4H2O salt in degassed water then mixing with EDTA in degassed water. The Fe/EDTA was removed from the Coy chamber for the Fenton reactions. rRNA was suspended to 0.5 μg μl−1 in 20 mM HEPES (pH 7.6), and 30 mM KCl, with 0% or 5% (v/v) glycerol and either 1 mM Fe/10 mM EDTA/10 mM ascorbate plus 0.3% (v/v) H2O2 or 10 mM EDTA as the reaction initiators wherein the initiators were separately dispensed onto the tube wall then vortexed with the other components. For the zero time points, reaction components were mixed in tubes containing the thiourea quenching agent at a final concentration of 100 mM. For non-zero time points, the reaction mixtures were prepared as bulk solutions and incubated at 37°C on a heat block, after which aliquots were removed at 0, 10 and 60 min and mixed with the thiourea quenching agent at a final concentration of 100 mM. The stopped solutions were immediately frozen and stored at −80°C. For analysis, samples were defrosted on ice, combined with 2X Gel Loading Buffer II, loaded onto a 1% Tris/borate/EDTA agarose gel and run at 120 V for 1.25 h.Purified rRNA from 2 or 25 mM FeCl2 . Reactions were placed on a 37°C heat block with aliquots removed at 0, 0.25, 0.5, 1, 2, 4 and 8 days for no M2+ and Mg2+ samples and at 0, 0.25, 0.5, 1 and 2 days for Fe2+ samples. Aliquots were immediately quenched with 100 mM final concentration sodium phosphate pH 7.6, centrifuged at 2,000 × g for 1 min, and the ApA-containing supernatant was collected to avoid transfer of Fe or Mg phosphate precipitate to the High Performance Liquid Chromatography (HPLC) column. The samples were stored at −80°C prior to placement in the HPLC where they were held at 4°C. HPLC analyses were conducted on an Agilent 1260 Infinity HPLC with a DAD UV–Vis detector, with a path length of 1.0 cm. Products of the reactions were separated using a Kinetex XB-C18 column . The flow rate was 0.3 ml min−1 and the column temperature was held at 25°C. The mobile phase was 100% 0.1% formic acid (aq) for 5 min ramped to 55% acetonitrile over 25 min. The acetonitrile concentration was then ramped to 100% and was held as such for 10 min before returning to 0.1% formic acid (aq) for column equilibration for 15 min. We recorded the elution at 210, 220 and 260 nm wavelengths, with a 180–400 nm spectrum detected in 2 nm steps. To characterize reaction products, standards were spiked into product mixtures. The spiked standards were 0.5 mM ApA in 20 mM HEPES (pH 7.6), 30 mM KCl, 5% (v/v) glycerol with either water, 2.5 μM adenosine, 3′-adenosine monophosphate or 2′,3′-cyclic adenosine monophosphate.In-line cleavage reagents were prepared as described above in the Coy chamber. In duplicate reactions, 0.5 mM ApA RNA dinucleotide  was suspended in degassed 20 mM HEPES/NaOH (pH 7.6), 30 mM KCl, 5% (v/v) glycerol and combined with either water, 25 mM MgCl2, and then incubated at 37°C for 2 days. The sample was analyzed by liquid chromatography mass spectrometry using an Agilent 1290 HPLC; Agilent 1260 Autosampler and DAD UV–Vis detector; path length: 0.6 cm; Agilent 1260 quaternary pump and RID; column: Phenomenex Kinetex 2.6 mmxB-C18100Å, LC column 150 × 2.1 mm; column temp: 25°C; 10 μl injection with needle wash, 100 μl s−1 injection speed. The solvents were (A) 0.1% formic acid in LC–MS grade water and (B) LC–MS grade acetonitrile with a flow rates of 0.3 ml min−1; gradient: 5 min 100% A, 0% B; 20 min ramp to 45% A, 55% B; 10 min 0% A, 100% B; 1 min ramp 100% A, 0% B; 9 min 100% A, 0% B. Elutions were recorded at 210, 220 and 260 nm, with the entire spectrum (180–400 nm) detected in 2 nm steps. This system was coupled to an Agilent 6130 single quad MS Electrospray Ionization Mass Spectrometry system with scanning of ±65 to ±2,000 m/z and capillary voltage of 2.0 kV.ApA was prepared anoxically at 0.5 mM with 20 mM HEPES pH 7.6, 30 mM KCl, 5% (v/v) glycerol and 25 mM FeCl−1 NaCl, 10 g l−1 tryptone, 5 g l−1 yeast extract) amended with 4 mM tricine, 50 mM sodium fumarate and 80 mM 3-(N-morpholino)propanesulfonic acid . Fifty ml cultures containing all of these ingredients plus 0.25% (v/v) glycerol were inoculated from glycerol stocks of E. coli MRE600 cells and shaken overnight at 37°C with or without O2 and with either 1 mM FeCl2 or ambient Fe2+ [6–9 μM, measured by the ferrozine assay . Elution buffers contained the same composition as the wash buffer except for NH4Cl (300 mM). Frozen cell pellets were resuspended in ribosome wash buffer and lysed in a BeadBug microtube compact homogenizer using 0.5 mm diameter zirconium beads (Benchmark Scientific). Cell lysate was transferred into centrifuge bottles inside the Coy chamber which were tightly sealed to prevent O2 contamination. Cell debris were removed by centrifuging outside of the Coy chamber at 30,000 × g for 30 min at 4°C. The soluble lysate was then transferred back into the chamber and loaded onto a column containing pre-equilibrated, cysteine-linked, SulfoLink™ Coupling Resin (Thermo Fisher Scientific). The resin was washed with 10 column volumes of wash buffer. Ribosomes were eluted into three 10 ml fractions with elution buffer. Eluted fractions were pooled inside the Coy chamber into ultracentrifuge bottles which were tightly sealed. Ribosomes were pelleted outside the chamber by centrifuging at 302,000 × g for 3 h at 4°C under vacuum in a Beckman Optima XPN-100 Ultracentrifuge using a Type 70 Ti rotor. Tubes containing ribosome pellets were brought back into the Coy chamber and suspended in buffer containing 20 mM HEPES (pH 7.6), 30 mM KCl and 7 mM β-mercaptoethanol, heat-sealed in mylar bags, and stored at −80°C. Ribosome concentrations were calculated with a NanoDrop spectrophotometer assuming 1A260 = 60 μg ribosome ml−1 (conversion factor provided by New England Biolabs). This conversion factor was used to estimate the molecular mass of bacterial ribosomes, from which molarity was calculated. Biological triplicates of each growth and purification method were taken for downstream analyses.The ribosome purification procedure was modified from Maguire et al. . All puret al.  as described in Bray and Lenz et al. .−1 in 2X SDS-PAGE loading buffer, heated at 95°C for 5 min, and then incubated on ice for 2 min. Samples were loaded onto a 12% SDS acrylamide gel with a 4% stacking gel and run at 180 V for 60 min.For SDS-PAGE, purified ribosomes were normalized to 3.33 mg mlet al. , 0.1 mM amino acid mix, 0.2 mM tRNAs, ∼0.2 μg μl−1 of dihydrofolate reductase mRNA, and 3 μl of factor mix  from the PURExpress® Δ Ribosome Kit (New England Biolabs). The reaction buffer was based on Shimizu et al. , 2 mM adenosine triphosphate (ATP), 2 mM guanosine triphosphate (GTP), 1 mM uridine triphosphate (UTP), 1 mM cytidine triphosphate (CTP), 10 mM creatine phosphate (CP) and 53 μM 10-formyltetrahydrofolate. Divalent cation salts (MgCl2 or FeCl2) were added to 9 mM final concentration. The reaction buffer was lyophilized and stored at −80°C until resuspension in anoxic nuclease-free water immediately before experiments in the Coy chamber. Reaction mixtures were assembled in the chamber and run at 37°C in a heat block for 120 min. Reactions were quenched on ice to terminate translation (+ by dihydrofolic acid (51 μM). Assays were performed by adding 5 μl of protein synthesis reaction to 995 μl of 1X assay buffer. The NADPH absorbance peak at 340 nm (Abs340) was measured in 15 s intervals over 2.5 min. The slope of the linear regression of Abs340 versus time was used to estimate protein activity (Abs340 min−1).Translation reactions were based on the methods of Bray and Lenz et al.  with minu et al. , with HEnslation  and storAfter ribosomal purification, samples were reduced with β-mercaptoethanol, and then alkylated with 14 mM iodoacetamide  for 30 minutes at room temperature in the dark. Alkylation was quenched with 5 mM dithiothreitol for 15 min at room temperature in the dark. Proteins were purified by the methanol/chloroform precipitation method and were then digested with trypsin in a buffer containing 5% acetonitrile, 1.6 M urea and 50 mM HEPES (pH 8.8) at 37°C with shaking overnight. The digestion was quenched with addition of trifluoroacetic acid to a final concentration of ∼0.2%. Peptides were purified by Stage-Tip  prior to6 AGC (automatic gain control) target in the Orbitrap cell was followed by up to 15 MS/MS scans in the Orbitrap cell for the most intense ions. Selected ions were excluded from further sequencing for 90 s. Ions with single or unassigned charge were not sequenced. Maximum ion accumulation time was 1,000 ms for each full MS scan, and 50 ms for each MS/MS scan.Peptides were dissolved in a solution containing 5% acetonitrile and 4% formic acid and loaded onto a C18-packed microcapillary column  by a Dionex WPS-3000TPL RS autosampler (Thermostatted Pulled Loop Rapid Separation Nano/Capillary Autosampler). Peptides were separated by a Dionex UltiMate 3000 UHPLC system (Thermo Scientific) using a 112-minute gradient of 4–17% acetonitrile containing 0.125% formic acid. The LC was coupled to an LTQ Orbitrap Elite Hybrid Mass Spectrometer (Thermo Scientific) with Xcalibur software (version 3.0.63). MS analysis was performed with the data dependent Top15 method; for each cycle, a full MS scan with 60,000 resolution and 1 × 10E. coli database from UniProt containing common contaminants using the integrated Andromeda search engine .The results were processed using Perseus software . In the 2+ due to its fixed oxidation state and inability to generate hydroxyl radicals. We confirm the expectation that Mg2+-mediated in-line cleavage reactions are not inhibited by anoxia or hydroxyl radical quenchers , which is known to quench hydroxyl radical and to inhibit hydroxyl radical cleavage , is increased with Fe2+ by ∼300 times for the 23S and by ∼200 times for the 16S. We demonstrate that although Fe2+ interacts in the same way as Mg2+ with RNA, the cleavage potency of Fe2+ is greatly enhanced.In sum, reactions with Mgntration ,39 so th2+ catalyzes non-oxidative in-line RNA cleavage, a series of cleavage reactions were performed on the dinucleotide ApA. The products of the reaction were characterized by HPLC via spiking with standards, and by LC-MS. A small RNA with only one possible cleavage site allowed us to identify specific cleavage products, which report on the mechanism of scission. In-line cleavage leads to a 2′,3′-cyclic phosphate upstream of the scission site and a downstream 5′-OH RNA fragment. Subsequently, the 2′,3′-cyclic phosphate can hydrolyze to either a 2′- or 3′- monophosphate. Conversely, oxidative cleavage of RNA by a hydroxyl radical that may be formed during iron oxidative processes abstracts a proton from a ribose sugar leading to a variety of products but not 2′,3′-cyclic phosphate, 2′-phosphate, or 3′-phosphate RNA fragments  and 3′-adenosine monophosphate (3′-AMP)  Figure . The rep2 and Fe2+ content during bacterial growth could affect the iron content of ribosomes. However, the vast majority of ribosomal M2+ ions are exchangeable . These results show that the vast majority of ribosomal M2+ ions are exchangeable and that M2+ exchange takes place during purification.We hypothesized that Oangeable  and cano buffers  to maintn Figure . All rib2+ ions are not exchangeable during purification. Ribosomes retain this subset of in vivo divalent cations after purification. We harvested E. coli in log phase from four growth conditions: oxic or anoxic with high Fe2+ in the medium (1 mM Fe2+), and oxic or anoxic without added Fe2+ in the growth medium (6–9 μM Fe2+). Ribosomes from E. coli grown in pre-GOE conditions  contained quantitatively reproducible elevated levels of Fe2+ after purification in solutions containing Mg2+. We detected around 9 mol Fe mol−1 ribosome from cells grown in pre-GOE conditions purified in solutions with high Mg2+ .A small subset of ribosomal M+ Figure . The thrin vitro under standard conditions (with 10 mM Mg2+) and also in 8 mM Fe2+ + 2 mM Mg2+ under anoxia. Regardless of whether translation activity was assayed in the presence of 10 mM Mg2+ or 8 mM Fe2+ + 2 mM Mg2+, ribosomes synthesized aerobically in the absence of Fe2+ have higher activity than the ribosomes synthesized anaerobically in the absence of Fe2+ . The absence of hydroxyl radical intermediates in the anoxic cleavage reaction is confirmed by the lack of inhibition by a hydroxyl radical quencher known to inhibit Fenton chemistry , andrates of in-line cleavage are significantly greater for Fe2+ but not Mg2+ confers oxidoreductase functionality to some RNAs 62+ is a stronger acid than Mg(H2O)62+; depletion of electrons is greater from water molecules that coordinate Fe2+ than from those that coordinate Mg2+. The lower pKa of Fe(H2O)62+ may promote protonation of the 5′-OH leaving group during cleavage. Metal hydrates with low pKas have been reported to induce RNA cleavage better than less acidic metal hydrates  during purification of ribosomes. Ribosomes purified in either Fe2+ or Mg2+ associate with 500–1000 M2+ ions that match the type of ion in the purification buffers. Our data support the tight association and lack of exchange of around 9 M2+ per ribosome. This subset of M2+ do not exchange during purification. The number of non-exchangeable M2+ closely matches the number of M2+ identified previously as a special class of deeply buried and highly coordinated M2+ in dinuclear microclusters (M2+-μc's)  . Mg2+ ior 10 M2+ . Displac or Fe2+ , dependi2+/Mg2+ ratios because of low intracellular Fe2+ availability and lability .The raw files are available at gkaa586_Supplemental_FileClick here for additional data file.


Sintered materials produced through the classical solid-state method, involving ball milling, show much larger grain sizes and slightly lower density than those obtained in samples produced from attrition-milled precursors. On the other hand, electrical resistivity has been drastically decreased, accompanied with a slight decrease in the Seebeck coefficient in samples obtained from these attrition-milled precursors. Moreover, the use of an attrition milling process leads to a very important reduction in processing time (around 75%), together with a slight power factor improvement of around 10%, when compared to the classically prepared samples.Attrition milling is an easily scalable and environmentally friendly process used to produce Ca For this type of application, it is necessary to produce materials with high TE performances, which are determined through ZT (TE figure of merit), involving the Seebeck coefficient (S), electrical resistivity (ρ), thermal conductivity (κ), and absolute temperature (T) parameters in the well-known expression, ZT = TS2/ρκ . This re2Te3 [xCoO2 [2Sr2Co2Ox or Ca3Co4O9+δ [Semiconducting and intermetallic materials as Bi3 [xCoO2 . These c3Co4O9+δ ,6. On th3Co4O9+δ materials using an easily scalable and environmentally friendly process, in a short processing time. Moreover, samples produced through the classical ceramic method will also be prepared to be used as reference.Taking into account these characteristics and previously published works about the effect of precursors sizes on the thermoelectric properties of ceramic materials , the aim3Co4O9+δ precursors were prepared using CaCO3  and Co3O4  commercial powders in the appropriate proportions. They were mixed thoroughly and milled using water media for 3 h in two different devices: attrition or ball mill. Attrition milling has been performed at 300 rpm using 1 mm diameter zirconia balls, while ball milling has been done with 10 mm diameter agate balls at 300 rpm using; in both cases, the milling media: precursor powder weight relationship is 10:1. These mixtures were used to produce the Ca3Co4O9+δ materials via attrition milling or through the classical ceramic route after sintering, respectively. After drying the different suspensions under infrared radiation, they were manually ground to obtain fine powders, which were subjected to different calcinations: a two-step process (750 and 800 °C for 12 h) in the classical ceramic route, previously reported as the optimal calcination conditions [3) under 400 MPa and sintered at 900 °C for 24 h in the classical solid-state route, and from 4 to 16 h at 900 °C for the attrition-milled precursors.Ca3Co4O9+δ phase appear.The precursors decomposition has been evaluated by DTA–TGA  between room temperature and 800 °C at 10 °C/min heating rate. Phases identification has been made through XRD  on the surface of sintered samples with CuKα radiation, and 2θ between 10 and 40 degrees, where the main diffraction peaks of Ca3 as a theoretical one [Microstructural observations were performed on precursor powders and longitudinal surfaces of sintered samples, using secondary electrons, in a field emission scanning electron microscope  fitted with an energy dispersive spectrometry (EDS) device, . Apparent density has been determined on several samples for each preparation conditions, taking 4.677 g/cmical one .Electrical resistivity and Seebeck coefficient were simultaneously determined using the standard four-probe configuration, in the steady state mode, using an LSR-3 system  between 50 and 800 °C under He atmosphere. With these data, PF has been calculated to determine the electrical TE performances.TGA analysis performed on the different dry precursors provides information about their weight evolution with temperature, giving information about the decomposition and phase formation processes, as shown in The first reaction is the main one in the calcination step, being responsible for the weight losses. On the other hand, the second reaction is happening, to a lower extent, during this calcination step, slightly increasing the samples weight. Consequently, it can be deduced that attrition-milled samples have higher reactivity than the ball-milled ones due to their smaller particle sizes, and the second reaction is produced in these precursors. Finally, the third reaction is mainly produced during the sintering step in both cases.3 decomposition energy, due to their much smaller particle sizes.DTA and TGA analyses have been simultaneously performed to evaluate the sign and magnitude of heat flow in the processes observed in the TGA graph. The sample evolution with temperature observed in the TGA analysis are confirmed through the DTA results displayed in These characteristics are illustrated in 3Co4O9+δ phase, in agreement with previous studies [3Co2O6+δ phase [Representative XRD patterns performed on the surface of sintered samples are displayed in  studies . Moreove studies . On the +δ phase  can be fRepresentative FESEM images of sample surfaces are shown in 3 for the samples prepared through the classical ceramic route, while it is between 3.50 ± 0.05 and 3.67 ± 0.03 g/cm3 for the ones produced from attrition-milled precursors, sintered for 4 and 16 h, respectively. Even with this enhancement of density values, they are still much lower than the produced through spark plasma sintering (4.35–4.58 g/cm3) [3) [3) [3) [3Co4O9+δ phase stability temperature (around 926 °C).The microstructural features previously discussed are reflected in the sample’s density, determined through Archimedes’ method, which is 3.43 ± 0.02 g/cm8 g/cm3) ,14, hot cm3) [3) ,16, or t [3) [3) . On the  [3) [3) . The obs [3) [3) , leading4+ to Co3+ [The evolution of electrical resistivity with temperature for all samples is shown in  to Co3+  and meta to Co3+ ,22,23. F to Co3+  or by di to Co3+ . On the  to Co3+ ,27,28. I2m), obtained in samples sintered for 12 h, are about 10% higher than the obtained through the classical ceramic route in this work, or in the literature (0.10–0.20 mW/K2m) [2m) [PF variation with temperature has been calculated for all samples, and the results are presented in  mW/K2m) ,23. On t mW/K2m) , or lase2m) [2m) . In spit3Co4O9+δ thermoelectric material has been presented. The use of attrition milling procedure has led to very reactive precursors due to their very small particle sizes, when compared to those obtained in the classical ceramic route. Smaller grain sizes and larger densities have been obtained with the attrition-milled precursors after sintering. Electrical resistivity has been drastically reduced in these attrition-milled samples, while Seebeck values have been only slightly decreased. Consequently, the highest power factor has been increased in around 10% at 800 °C for attrition-milled samples, when compared to the classical solid state sintered ones. Finally, the use of attrition-milled precursors has allowed a drastic reduction in processing time, together with a slight increase in thermoelectric performances.In this work, a simple, economic, easily scalable, and environmentally friendly process to produce the Ca


E4, E5a, and E5b mRNAs accounted for 96% of the expression of 9 mRNAs. The alteration of viral DNA load during recurrence paralleled the mRNA expression levels, and the expression of all mRNAs showed a similar curve. E4, E5a, and E5b were expressed in the middle to upper part of the epithelium and were co-expressed in the same cells. E4 immunohistochemistry demonstrated an extensively positive reaction in the upper cell layer in accordance with E4 mRNA expression. These results suggest that individual viral genes are coordinately expressed for viral replication, virus release, and immunosurveillance avoidance. The newly developed E4-specific monoclonal antibody can be applied to further functional studies and clinical applications such as targeted molecular therapies.Laryngeal papilloma (LP) associated with human papillomavirus (HPV)-6 or -11 infection shows aggressive growth. However, the detailed molecular mechanism of virus-driven tumorigenesis has not been uncovered fully. HPV-6 viral gene expression and dynamic alterations were investigated with in situ localization of viral DNA and RNA in 13 patients with HPV-6-infected laryngeal papilloma. The average viral load was 4.80 × 10 Laryngeal papilloma (LP) is the most common benign epithelial tumor of the larynx, appearing as finger-like projections or multiple fronds of stratified squamous epithelium. The typical pathological findings are hyperplasia of basal cells and large vacuolated epithelial cells with clear cytoplasm. The estimated incidence of LP is approximately 4 per 100,000 in children and 2 per 100,000 in adults ,4. FrequE1, E2, E4, E5, E6, and E7), 2 late regions (L1 and L2), and the long control region (LCR). According to their association with cancer, HPV is divided into low-risk and high-risk types. HPV-6 and -11, observed in LP, are classified as the low-risk type. HPV preferentially infects mitotically active basal cells through microtears in the mucosal and cutaneous epithelium [E1 and E2) and packaging (L1 and L2) with greater diversity in the remaining genes , which have roles in driving cell cycle entry, immune evasion, and virus release [HPVs are small double-stranded DNA viruses that infect mucosal and cutaneous epithelia and cause many tumorous lesions from benign warts to cancer of the anogenital and otolaryngological regions . The app release . Althoug release , they re release . E4 is located centrally within the E2 gene (E4 gene product (E1^E4) is translated from a spliced mRNA [E4 transcripts have been reported, it is the product of the abundant E1^E4 mRNA that has been most extensively analyzed [E4 protein encoded by HPV-16 appears to have the ability to bind to keratin and to reorganize the cellular keratin network, suggesting a role in virus release and transmission [E5a, which is called E5b. E5b shows no sequence similarity to other E5 proteins. Although HPV-16 E5 can activate the epidermal growth factor receptor, inhibit the expression of the p21 tumor suppressor gene [E5a and E5b in low-risk HPVs are not understood fully.  E2 gene , and theced mRNA . Althouganalyzed . E4 protsmission . HPV-6 asor gene ,13, and sor gene , the bioE6, E7, E1, E2, E4, E5a, E5b, L2, and L1 mRNAs, an mRNA in situ hybridization (ISH) method for E6, E2, E4, E5a, and E5b, and created an anti-E4 monoclonal antibody for immunohistochemical localization of E4 in LP.LP caused by HPV-6 and -11 infection is a chronic and recurrent disease that significantly lowers patients’ quality of life and may lead to life-threatening conditions. Furthermore, effective clinical treatments have not been established. Therefore, it is vitally important to characterize the underlying pathogenic mechanisms. However, the detailed molecular mechanisms of HPV-driven tumorigenesis are mostly unknown. In the present study, we established a real-time polymerase chain reaction (PCR) assay to measure the absolute levels of The subjects consisted of 16 patients with LP aged from 4 to 67 years at the first visit to our hospital between 2010 and 2018. Tumor specimens were frozen immediately in liquid nitrogen at biopsy or surgical excision between 2010 and 2018 and stored at −80 °C until analysis. The HPV subtypes were determined as described previously . Of the DNA was extracted from fresh-frozen samples using a Gentra Puregene Tissue Kit  . The preL1 region. When no PCR amplification occurred with the GP5+/GP6+ or MY09/MY11 primers, 10-fold diluted first-round PCR products were used as template DNA for nested PCR using the GP5+/GP6+ primer pair. PCR products of the expected size  were purified and directly sequenced with an ABI PRISM 3130xl Genetic Analyzer . The sequences were aligned and compared with the L1 gene of known HPV subtypes in GenBank using blastn: https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome (accessed on 13 February 2021).Negative (water) and positive (DNA from an HPV-16-positive CaSki cell line) controls were included in each amplification series. To determine the HPV subtypes in the samples, we used the degenerate consensus primer sets GP5+/GP6+ and MY09/MY11 [L1 to E6 of HPV-6a (GenBank accession no. L41216), HPV-6b (X00203), and HPV-6vc (JN252316). For amplification, PCR was performed with LCR-F and LCR-R primers  and 10 ng genomic DNA. The PCR profile was as follows: denaturation at 95 °C for 15 min, followed by 45 cycles at 95 °C for 15 s, 60 °C for 30 s, and 72 °C for 2 min, and a final extension at 72 °C for 5 min. PCR fragments of the expected size were purified by the Wizard SV Gel and PCR Clean-Up System . The purified PCR products were cloned into the pGEM-T Easy vector  and sequenced with an ABI PRISM 3130xl Genetic Analyzer . To identify the HPV-6 subtype, we designed LCR-degenerate primers in the conserved region from The obtained nucleotide sequences of the LCR region were compared with HPV-6a, HPV-6b, and HPV-6vc by the GENETYX sequence analysis software package . Multiple alignments for phylogenetic analysis were performed by the GENETYX sequence analysis software package. A phylogenetic tree was constructed by the unweighted pair group method with arithmetic mean, using the GENETYX sequence analysis software package. The LCR region sequence of HPV-11 served as an outgroup for the phylogenetic tree.E6 DNA copy number in genomic DNA was determined with the ABI Prism 7300 Sequence Detection System  or CFX96 Touch™ Real-Time PCR Detection System . Briefly, serially diluted HPV-6 E6 plasmid  was used as a standard (1.0 × 101–1.0 × 107 copies/2 μL) to establish a standard curve. p1478 HPV-6 E6 was a gift from Peter Howley . The genomic DNA samples from the patients were diluted to 30 ng/2 μL and used for measurement. The PCR reaction mixture (10 μL) contained 0.2 μM primers, 0.15 μM TaqMan probe . To establish real-time PCR assays to measure the absolute levels of E7, E1, E2, E4, E5a, E5b, L2, and L1 mRNAs, we cloned 4 regions of HPV-6 gene fragments using genomic DNA from patient 3  using primer pairs  using primer pairs  using primer pairs  using primer pairs . The PCR conditions comprised: 95 °C for 15 min, followed by 40 cycles at 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 3 min, and finally 72 °C for 7 min. The PCR fragments of the expected size were purified using the Wizard SV Gel and PCR Clean-Up System . The purified PCR products were cloned into the pGEM-T Easy vector  and sequenced using an ABI PRISM 3130xl Genetic Analyzer. The sequences obtained were analyzed as mentioned above. The plasmids  were serially diluted to 2.0 × 101–2.0 × 107 copies/2 μL as standards for E7, E1, E2, E4, E5a, E5b, L2, and L1 genes to establish a standard curve in real-time PCR. Real-time PCR was performed with the ABI Prism 7300 Sequence Detection System  or CFX96 Touch™ Real-Time PCR Detection System . The PCR reaction mixture (10 μL) contained 0.2 μM primers, 5 μL SYBR Premix Ex TaqTM II , and 2 μL standard plasmid DNA (2.0 × 101–2.0 × 107 copies) or cDNA. The PCR profile was as follows: 95 °C for 30 s followed by 40 cycles at 95 °C for 5 s and 60 °C for 30 s. Specific amplification of each cDNA was verified by melting curve analysis and gel electrophoresis of the products. The real-time PCR assay for E6 mRNA was performed with the same E6 primers and TaqMan probe as well as the same protocol described above. We also measured β-actin mRNA as the internal reference. The plasmid pCAG-mGFP-Actin (a gift from Ryohei Yasuda), carrying the entire coding region of β-actin , was serially diluted to 1.0 × 101–1.0 × 107 copies/2 μL to establish a standard curve. The PCR reaction mixture (10 μL) contained 0.2 μM primers, 0.15 μM TaqMan probe . Total RNA (500 ng) from each sample was reverse-transcribed using the PrimeScriptan probe , 5 μL Ta2O2 in methanol for 20 min. A drop of the HPV probe was added to the section, and a coverslip was applied. The probe and target DNA were denatured by incubating the slides at 92 °C for 5 min. After denaturation, the slides were transferred to a humidified chamber for hybridization at 37 °C for 16 h. After hybridization, the coverslips were removed, and the slides were bathed in Tris-buffered saline containing 0.05% Tween 20 (TBST). The coverslips were washed using GenPoint Detection system stringent wash solution  at 48 °C for 30 min, following by rinsing in TBST. Detection of the hybridized probe was performed using the GenPoint Detection system with primary streptavidin-horseradish peroxidase (HRP), biotinyl tyramide, secondary streptavidin-HRP, and 3-3′-diaminobenzidine . The slides were counterstained with hematoxylin.Biotinyl tyramide-based ISH was performed using the HPV Types 6/11 Biotinylated DNA Probe  and the GenPoint tyramide signal amplification system for biotinylated probes according to the manufacturer’s protocol . The biotinylated DNA probe has been found to react with HPV-6 and -11 in formalin-fixed paraffin-embedded (FFPE) sections by ISH. Serial 4-µm-thick sections of FFPE samples were deparaffinized in xylene and rehydrated using a graded alcohol series. Target HPV DNA retrieval was performed in 10 mM sodium citrate (at pH 6.0) at 95 °C for 40 min followed by a 20-min cooldown period. The slides were digested with proteinase K  for 10 min at room temperature. Endogenous peroxidases were blocked with 0.3% HE6, E2, E4, E5a, and E5b genes of HPV-6 were amplified by the following PCR method. The primer sets are shown in E6; clone B plasmid for E2, E4, and E5a; clone C plasmid for E5b), 0.24 μM forward and reverse primers, and 6.3 μL GoTaq® Green Master Mix . The PCR conditions comprised: 95 °C for 5 min, followed by 35 cycles at 95 °C for 30 s, 60 °C for 30 s, and 72 °C for 1 min, and finally 72 °C for 5 min. The PCR products were subcloned into the pGEM-T Easy vector  as described above. Anti-sense RNA probes were transcribed from linearized plasmids using a digoxigenin (DIG)-labeling mix  and SP6 or T7 RNA polymerase , and digested with DNase I . A total of 1 μg of the linearized plasmid DNA was used in a 20-μL reaction for DIG-RNA labeling. The probes were stored at −80 °C until use. For RNA-ISH, the ®; Matsunami Glass, Osaka, Japan). The sections were deparaffinized in xylene and rehydrated in a graded alcohol series, followed by incubation in ultra-pure water for 5 min. After washing with phosphate-buffered saline (PBS) with 0.1% Tween 20 (PBST), the sections were incubated in PBST including proteinase K  for 5 min at 37 °C, and fixed in 4% paraformaldehyde  for 15 min at room temperature. After washing with PBST, pre-hybridization was performed in a hybridization buffer containing 50% deionized formamide, 5× saline-sodium citrate , 50 µg/mL heparin , 100 µg/mL Escherichia coli tRNA , 1% sodium dodecyl sulfate (SDS), and 0.1% Tween 20 at 60 °C for 1 h, and hybridization was performed with DIG-labeled RNA probes (1 µg/mL in hybridization solution) at 60 °C overnight in a humidified chamber. After hybridization, the sections were washed as follows: for 5 min in 50% formamide in 2× SSC with 0.1% Tween 20 at 60 °C; 5 min in 2× SSC at 60 °C; and 5 min in 0.2% SSC at room temperature. The sections were washed 3 times with PBST at room temperature, incubated with blocking buffer  for 15 min, and incubated with 1:3000 anti-Digoxigenin-AP , Fab fragments  in the blocking buffer at 4 °C overnight. The sections were rinsed 3 times with PBST and washed with reaction buffer . Section detection was carried out using a nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate solution  in the reaction buffer. The sections were washed 3 times with PBST and counterstained with Vector® Nuclear Fast Red . LP FFPE samples were sectioned at 4 μm and mounted onto adhesive glass slides  and TSATM Cyanine 5 System . An anti-sense E4 DIG-RNA probe was prepared as described above. Anti-sense E5a and E5b biotin probes were transcribed from linearized plasmid using Biotin RNA Labeling Mix  and SP6 or T7 RNA polymerase, and digested with DNase I as described above. The probes were stored at −80 °C until use. Double fluorescence RNA-ISH was performed using the TSAE4 DIG-labeled RNA probe and anti-sense E5a biotin-labeled RNA probe (each 1 µg/mL in hybridization solution) or anti-sense E4 DIG-labeled RNA probe and anti-sense E5b biotin-labeled RNA probe (each 1 µg/mL in hybridization solution) at 60 °C overnight in a humidified chamber. After hybridization, the sections were washed as follows: for 5 min in 50% formamide in 2× SSC with 0.1% tween at 60 °C; 5 min in 2× SSC at 60 °C; and 5 min in 0.2% SSC at room temperature. The sections were washed 3 times with PBST at room temperature, incubated with blocking buffer (0.5% blocking reagent in PBST) for 15 min, and incubated with 1:100 anti-Digoxigenin-POD (horse-radish peroxidase), Fab fragments  in the blocking buffer at 4 °C overnight. The sections were rinsed 3 times with PBST and washed with Tris-NaCl-Tween buffer . The fluorescein tyramide reagent from the TSATM Fluorescein System  was diluted at 1:50 with the amplification diluent and mounted onto adhesive glass slides at room temperature for 10 min. Then, the sections were washed 3 times with PBST at room temperature. Endogenous peroxidases were blocked with 3% H2O2 in PBST at room temperature for 1 h. The sections were rinsed 3 times with PBST and incubated with Tris-NaCl-blocking buffer . Streptavidin-HRP was diluted at 1:250 with TNB buffer  and mounted on the slides for 30 min. The slides were washed 3 times with TNT buffer. The cyanine 5 tyramide reagent was applied to the slides for 10 min. Then, the slides were washed 3 times with TNT buffer and washed twice with PBS. Nuclei were counterstained with 10 µg/mL Hoechst 33,342  in PBST for 10 min. The slides were washed 3 times with PBS. Fluorescence was detected with an Axio Imager M2 microscope  equipped with Colibri.2 light-emitting diode (LED) illumination and the Apotome.2 system .The process from sectioning to hybridization was the same as described above. Hybridization was performed with an anti-sense VHH gene was fused with the mouse IgG Fc gene. The peptides and antibody were prepared by RePHAGEN Co., Ltd. .To generate a specific antibody against HPV-6 E1^E4, an alpaca variable domain of heavy chain of heavy-chain (VHH) antibody library was used as described previously . Then, tPlasmid Construction. HPV-6 E1^E4 and HPV-11 E1^E4 were cloned into the pcDNA3.1+ vector, with the 3× FLAG-tag at the N-terminus [CMTM7) tagged with 3× FLAG at the N-terminus was also used as described previously [terminus . Human Ceviously .Cell Culture and Transfection. Human embryonic kidney HEK293T cells were cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin. HEK293T cells (2.0 × 106 cells) were cultured in a 10-cm dish for 24 h. Immediately before transfection, the cells were rinsed and supplemented with fresh culture medium (high-glucose DMEM) without FBS and penicillin/streptomycin. Transfection was performed using HilyMax . HPV-6 E1^E4-, HPV-11 E1^E4-, and human CMTM7-expressing plasmids (30 µg) were mixed with 15 µL HilyMax and 900 µL high-glucose DMEM and incubated for 15 min. Then, the DNA mixture was added to the cells. After 4-h incubation, high-glucose DMEM including 10% FBS and penicillin/streptomycin was added to the cells. The cells were cultured for 48 h after transfection. Non-transfected cells were included in the transfection experiments.Western Blotting for HPV-6 E4. HEK293T cells expressing HPV-6 E1^E4, HPV-11 E1^E4, or human CMTM7 and non-transfected HEK293T cells were lysed in a buffer containing 62.5 mM Tris-HCl (pH 6.8), 2% SDS, and 10% glycerol. Lysate protein concentration was measured using a DC Protein Assay Kit . Each protein sample was prepared at 50 µg/10 µL in 62.5 mM Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, 6% mercaptoethanol, and 0.01% bromophenol blue and boiled for 5 min. The samples were electrophoresed in 12.5% acrylamide SDS-PAGE and transferred to polyvinylidene difluoride membranes. To confirm the expression of HPV-6 E1^E4, HPV-11 E1^E4, and human CMTM7, FLAG protein was detected with an HRP-conjugated monoclonal anti-FLAG M2 antibody  diluted at 1:1000 with Can Get Signal Solution 2 . To evaluate the specificity of the anti-HPV-6 E1^E4 antibody , the antibody was used as a primary antibody without dilution. A secondary antibody, goat IgG-conjugated with HRP against mouse IgG (H+L) , was used at a dilution of 1:1000 with Can Get Signal Solution 2 . Bands were visualized using Clarity Western ECL Substrate  and the ChemiDoc XRS+ System . E1^E4 immunohistochemistry, 4-μm-thick sections from FFPE samples were deparaffinized in xylene and hydrated in a graded alcohol series. Epitope retrieval was achieved by heating at 100 °C for 10 min in 1 mM Ethylenediaminetetraacetic acid (EDTA) buffer (pH 8.0). Endogenous peroxidase activity was quenched by incubating the sections in 0.3% H2O2 in methanol for 20 min at room temperature. According to the manufacturer’s protocol, a SAB-PO Kit  was used to detect immunoreactivity to HPV-6 E1^E4. After blocking non-specific reactions by incubation in 10% goat serum, the tissue slides were incubated with the primary antibody against HPV-6 E4  overnight at 4 °C. Subsequently, a biotin-labeled secondary antibody and peroxidase-labeled streptavidin were applied. Immunolabeling was visualized by incubation in 3-3′-diaminobenzidine, and stained slides were counterstained with hematoxylin.For HPV-6 p < 0.05. Statistical analyses were performed using NCSS Statistical Software ver.12 .Viral mRNA levels  were analyzed by the Kruskal–Wallis test, followed by the Dwass–Steel–Critchlow–Fligner multiple comparison test to detect statistically significant differences in the mRNA levels of the 9 viral genes. The correlations among the levels of the 9 viral mRNAs were analyzed by Spearman’s rank-order correlation test. The correlations between viral load and viral mRNA levels were analyzed as described above. Statistical significance was set at This study was approved by the Institutional Review Board of the University of Ryukyus and was carried out in accordance with the 1975 Declaration of Helsinki, as revised in 2008. Informed consent was obtained from all patients before enrollment.5 ± 1.86 × 105 copies/ng DNA  was the highest among these 9 genes  and was followed by E5b and E5a mRNA levels . E4 mRNA expression was the highest among the 9 viral genes in 8 of 10 patients, while E5a and E5b mRNA levels were higher than E4 mRNA expression in patients 2 and 3 . When the total expression of the 9 viral mRNAs was set to 100%, the percentage of E4, E5a, and E5b expression was 96% . The expression levels of the 9 viral mRNAs were determined in 19 tissues from 10 patients . E4 mRNA 2 and 3 a. MeanwhThe correlations of the expression levels of each mRNA with the other viral mRNAs are shown in E5a and L2 mRNA levels , and also tended to show positive correlations with E1 and L1 mRNA levels, although these relationships did not reach statistical significance . In contrast, there was no correlation between viral DNA load and the mRNA levels of E6, E7, E2, E4, and E5b .The correlations between viral DNA load and the levels of the 9 viral mRNAs were also examined by Spearman’s rank-order correlation . Viral DViral load and the expression of the 9 viral mRNAs were measured in case 10, who undertook 10 revision surgeries over a period of 80 months. The levels of the 9 viral mRNAs and viral load altered synchronously with each other’s peaks at 0, 35, and 71–73 months, with the lowest levels at 25 and 63 months . The shaE4, E5a, E5b, E6, and E2 signals were observed in 91.7% (11/12), 75% (9/12), 50.0% (6/12), 41.7% (5/12), and 41.7% (5/12) of LP cases, respectively  and E5a  mRNAs had a similar distribution pattern, and the merged image of E4 and E5a showed that they were co-expressed in LP , but not to HPV-11 E1^E4–3× FLAG fusion protein, human CMTM7-3× FLAG fusion protein, or lysate proteins from HEK293T cells .Immunohistochemistry for HPV-6 pression a,b. In cantibody c,d, but According to a recent investigation of the global genomic diversity of HPV-6, HPV-6a belongs to sublineage B3, HPV-6b is lineage A, and HPV-6vc is sublineage B1 . In 176 E4, E5a, and E5b mRNAs were exclusively expressed in the present study and represented 96% of the total expression of 9 HPV-6 mRNAs. The results suggest that these 3 genes are the most important for the HPV-6 life cycle. E4 protein, which has significant sequence heterogeneity between HPV types, may play an essential role in the virus escape step from the epithelial surface and reflects the different tropisms and transmission routes of different papillomaviruses [E4, E5a, and E5b were expressed in the middle to upper part of the epithelium and were co-expressed in the same cells, as observed using double fluorescence RNA-ISH. These results suggest that E4, E5a, and E5b work cooperatively for viral replication, virus release, and immunosurveillance avoidance. Although information for the DNA-ISH probe is not available, the DNA-ISH results in the present study showed that the cells expressing HPV-6 DNA were located in all LP layers, especially the middle to upper part of the tumor, in accordance with E4, E5a, and E5b mRNA expression. This observation also supports the above assumption related to E4, E5a, and E5b. Therefore, E4, E5a, and E5b, showing predominant expression in HPV-6-associated LP, are good candidates as therapeutic molecules in LP.aviruses ,24. E4, E1, E6, E7, L1, and L2 mRNA expression and viral load [E1, E6, E7, L1, and L2 were much lower than those of E4 and E5 in the present study. These differences need to be clarified in the near future.Recently, the raft culture method of LP was reported, and there was no significant relationship between ral load . AlthougE4 and E5 reported that E4 could destroy HPV-infected cells, and E5 has a role in viral DNA synthesis and escape from immune surveillance [E5 is a small, membrane-bound, highly hydrophobic protein, and the E5 gene is conserved in many HPVs [E5 has been investigated primarily in HPV-16, and it plays a vital role in the HPV life cycle by delaying normal epithelial cell differentiation, while maintaining cell cycle progression, enhancing the oncogenic ability of the significant transforming protein E7 [E5a and E5b in HPV-6 remain unclear. Although the expression levels of the 9 viral mRNAs were correlated with each other in the present study . In the high-risk types of HPV, E4 protein assembles into amyloid fibrils that can disrupt the keratin network and compromise the normal assembly of the cornified envelope [E4 amyloid fibers in low-risk HPV may contribute to virion release from the upper epithelial layers, and therefore affect infectivity and transmission [E4 immunohistochemistry demonstrated an extensively positive reaction in the upper cell layer in LP, in accordance with E4 mRNA expression. These results suggest that E4 has a role in virus release from LP and contributes to viral transmission. All LP samples with HPV-6 infection showed a strong positive reaction in the upper cell layer. These findings suggest that the E4 immunohistochemistry can be used for the detection of HPV-6 infection. Since the functional role of E4 is not understood fully, this antibody will help in the analysis of neoplastic development and the viral life cycle in low-risk HPV.An anti-HPV-6 envelope . Althougsmission . E4 immuE4, E5a, and E5b mRNAs accounted for 96% of the expression of the 9 mRNAs. The alteration of viral DNA load during recurrence paralleled the mRNA expression levels, and the expression of all mRNAs showed a similar curve. E4, E5a, and E5b were expressed in the middle to upper part of the epithelium and were co-expressed in the same cells. E4 immunohistochemistry demonstrated an extensively positive reaction in the upper cell layer in accordance with E4 mRNA expression. These results suggest that individual viral genes are coordinately expressed for viral replication, virus release, and immunosurveillance avoidance. However, there was no significant correlation between clinical parameters and viral load/HPV mRNA expression levels.In the present study, we examined the HPV DNA viral loads, expression levels of 9 viral mRNAs, and cellular distribution of mRNA expression in 13 LP patients with HPV-6 infection .


The mechanochemical reaction between Pr6O11, BaO2, and MnO powders with cationic stoichiometric ratios of 1/1/2 for Pr/Ba/Mn was performed using high-energy milling conditions in air. After 150 min of milling, a new phase with perovskite structure and cubic symmetry consistent with the A-site disordered Pr0.5Ba0.5MnO3 phase was formed. When this new phase was subsequently annealed at a high temperature in an inert Ar atmosphere, the layered PrBaMn2O5+δ phase was obtained without needing to use a reducing atmosphere. At 1100 °C, the fully reduced layered PrBaMn2O5 phase was achieved. A weight gain was observed in the 200–300 °C temperature range when this fully reduced phase was annealed in air, which was consistent with the transformation into the fully oxidized PrBaMn2O6 phase. The microstructural characterization by SEM, TEM, and HRTEM ascertained the formation of the intended PrBaMn2O5+δ phase. Electrical characterization shows very high electrical conductivity of layered PBMO in a reducing atmosphere and suitable in an oxidizing atmosphere, becoming, therefore, excellent candidates as solid oxide fuel cell (SOFC electrodes).In this work, a mechanochemical route was proposed for the synthesis of the PrBaMn Solid oxide fuel cells (SOFCs) are one of the most promising and environmentally friendly technologies for generating electricity from a variety of fuels. SOFCs directly transform chemical energy into electricity without the need for combustion , resulting in much higher efficiencies, since losses derived from thermodynamic considerations are minimized ,2. If hy3) have been proposed as candidates for anodes, as they are flexible in composition and allow a high degree of cationic substitution in A- and B-sites, which permits the tailoring of the final properties, such as the electronic and ionic conductivities, by properly adjusting the stoichiometry. The presence in the B-site of transition metals with multiple oxidation states is of primary importance because it largely determines the electrocatalytic properties and stability upon redox cycling [This is one of the reasons why the search for alternative anode materials resistant to carbon and sulfur is receiving special and continuous attention . Oxides  cycling ,11. The  cycling ,13, in w2BB′O6, such as Sr2CoMoO6-δ, Sr2MgMoO6-δ, or Sr2TiMoO6-δ [2O6), and particularly double layered manganites, including PrBaMn2O5+δ (PBMO), have been proposed as promising alternatives to Ni/YSZ, not only as potential anode materials in SOFCs [Double perovskites with the general formula ATiMoO6-δ ,15,16, hin SOFCs , but alsin SOFCs , due to in SOFCs .4+/Mn3+/Mn2+), which favor the intake and release of oxygen within the vacancy rich Pr A-site layer during the redox processes.In PBMO structure, the Ba and Pr cations are not distributed randomly in the perovskite A-site, but ordered in alternating Pr and Ba layers along (001). All the oxygen vacancies in PBMO are located in the Pr A-site layers , which p2 [0.4Ce0.6O2-δ (LDC)/La0.9Sr0.1Ga0.8Mg0.2O3-δ (LSGM)/NdBa0.5Sr0.5Co1.5Fe0.5O5+δ-Ce0.9Gd0.1O2-δ (NBSCF50-GDC), a maximum power density of 0.66 W/cm2 was obtained at 800 °C in humidified H2 (3% H2O) [2 were obtained at 850 °C in humidified H2 when PBMO with a 15 wt.% Co-Fe catalyst was used, with no observable degradation in H2S/H2 [2 at 850 °C [PBMO has shown an electrical conductivity of 8.16 S/cm at 800 °C in 5% H2 . In an e(3% H2O) . An LDC (3% H2O) ,24. Althn H2S/H2 . This sat 850 °C . The exst 850 °C .0.5Ba0.5MnO3 precursor is obtained from a sol-gel route using EDTA or citric acid as the chelating agent, which includes a final pyrolysis step at high temperature in air [0.5Ba0.5MnO3 precursor is generally composed of a mixture of two phases with cubic and hexagonal structures. The major cubic phase corresponds to the A-site disordered perovskite, while the minor hexagonal phase is associated with the BaMnO3 secondary phase, whose formation is favored by a high extent of oxidation from Mn3+ to Mn4+ under the experimental conditions of synthesis. In a second step, the Pr0.5Ba0.5MnO3 precursor is annealed at ~800–900 °C in a reducing atmosphere, during which the hexagonal structure is suppressed, and the A-site ordered layered PBMO is formed with a tetragonal symmetry.PBMO powders are generally prepared by a two-step synthesis procedure. First, a disordered Pre in air ,25,26,27e in air . Note thMechanochemistry has shown to be an efficient, reproducible, and relatively simple way to obtain mixed oxides with simple perovskite structure that can be used as components in SOFCs ,31,32,33n+1BnO3n+1), Aurivillius (Bi2O2) (An−1BnO3n+1) or double perovskites (A2BB′O6) phases, but in general, an annealing step after the milling treatment of the reactant mixture was required to obtain the intended final layered phase [0.92Co2O6-δ-Ce0.8Sm0.2O1.9, from the different phases previously synthesized by other methods [2O5+δ and BaSmMn2O5+δ previously obtained at high temperatures [Several studies can be found in the literature dealing with the mechanochemical synthesis of selected layered perovskite oxides, such as the Ruddlesden-Popper , barium peroxide  and manganese (II) oxide  powders were used as the starting reactants for the mechanochemical synthesis of PrBaMn2O5+δ. For the experiments, the stoichiometric amounts of the powder reactants to produce 5 g of PrBaMn2O5+δ and six tungsten carbide (WC) balls (d = 15 mm and m = 26.4 g) were placed in a 60 mL tempered steel vial and milled in air in a planetary mill  at a spinning rate of 600 rpm. The milling process was stopped at 15 min intervals for X-ray diffraction (XRD) inspection. A total milling time of 150 min was used.Praseodymium  oxide  equipped with a θ/θ goniometer, a graphite-diffracted beam monochromator and a solid-state detector (X’Cellerator). The diffraction patterns were acquired using Cu Kα radiation from 10° to 140° (2θ) in step-scan mode with a step size of 0.05° and a counting time of 300 s/step. The diffraction line positions were corrected with silicon powder . Phase quantification, lattice parameters, crystalline domain sizes (D) and microstrains (e) were calculated from the Rietveld refinement tool offered by the X’Pert HighScore Plus software  using pseudo-Voigt functions to describe the line profiles.6 filament (point resolution = 0.25 nm). HR micrograph analysis, fast Fourier transform (FFT), and phase interpretation were performed with the Gatan Digital Micrograph software (Gatan Inc.) and the Java version of the electron microscope software (JEMS).For the microstructural characterization, the powder samples were dispersed in acetone and droplets of the suspension were deposited onto a holey carbon copper grid. The scanning electron microscopy (SEM) images were obtained on a Hitachi S-4800 SEM-FEG microscope in secondary electron mode at an acceleration voltage of 5 kV. Energy-dispersive X-ray analysis (EDX) was performed with a detector coupled to the SEM microscope using an acceleration voltage of 20 kV. The transmission electron microscopy (TEM) and high-resolution (HRTEM) images, selected area electron diffraction (SAED) patterns, and EDX spectra were taken on a 200 kV JEOL-2100-PLUS microscope equipped with a LaBThe thermogravimetric measurements were carried out in a simultaneous thermal analysis equipment  on 60–80 mg of powders using an alumina sample pan at a heating rate of 10 °C/min from room temperature up to 1100 °C in two different flowing atmospheres, air (50 mL/min) and Ar (50 mL/min).2O5+δ were measured using an impedance analyzer model Solartron 1260A at open circuit voltage (OCV). All measurements were made at from 800 to 100 °C, with a range of 100 °C. The specimens were measured in synthetic airflow (150 mL/min) and Ar/10%H2 (150 mL/min) with a frequency range of 1 MHz–0.1 Hz. All the spectra were analyzed with the ZView software. Platinum wires were used as voltage and current collectors in a single chamber configuration. For the impedance measurements, 0.6 g of the powder was compacted in a disk shape (∅ = 12 mm and thick = 1 mm) using a uniaxial press at 100 MPa for 5 min and further sintered at 1500 °C in argon for 12 h, at heating and cooling rates of 5 °C/min, in a tubular furnace. Finally, the specimens were ground and polished to achieve a final thickness of 1 mm.Impedance spectra of reduced and oxidized layered PrBaMn6O11/BaO2/MnO powder mixture with a stoichiometric ratio according to Equation (1) was milled using the experimental conditions described in the Materials and Methods section.A Pr2) was considerably reduced after only 15 min of milling. Moreover, the XRD peaks also suffered from a large broadening. Both effects were the result of the high-intensity milling regime employed. At this short time of 15 min, new XRD peaks began to develop suggesting the formation of a new phase with a perovskite structure. With increasing milling time, the mechanochemical reaction progressed and the amount of this new phase continuously increased. After 150 min, the conversion was finished and the mechanochemical solid state reaction seemed to be nearly complete (from now this sample will be named PBMO-m). Only the existence of a minor amount of BaCO3, which was already present in the starting BaO2 as a minor phase, was observed. The percentage of the newly formed perovskite phase as a function of the milling time calculated from the XRD patterns in The milling process was stopped every 15 min and the powder was inspected by XRD. D = 14.3 nm) and the presence of a high number of defects  as a result of the high-intensity milling conditions employed. The XRD pattern was consistent with the A-site disordered Pr0.5Ba0.5MnO3 phase with cubic symmetry and space group Pm-3m (a = 3.8962(3) Å); the corresponding (h k l) are marked in a = 5.522(1) Å and c = 7.757(3) Å were obtained from this XRD pattern (PBMO-m sample), assuming the tetragonal structure with space group P4/nmm characteristic of the reduced-form of the PrBaMn2O5+δ phase. Furthermore, the presence of the hexagonal phase associated with the BaMnO3 secondary phase, as generally found in thermal-related synthesis processes, was not detected in this mechanochemical route.The XRD peaks of the new perovskite phase show extremely large broadening due to the small size of the coherent diffraction domains  was already well defined. The lattice parameters determined for this sample, labelled as PBMO-m + 900 °C/Ar, were a = 5.6179(4) Å and c = 7.7695(7) Å. Annealing at higher temperature (1100 °C) did not produce any new significant modification  and the sample, labelled as PBMO-m + 1100 °C/Ar, maintained similar lattice parameters (a = 5.6219(5) Å and c = 7.7584(1)), which were in close agreement with the literature values determined for the fully reduced PrBaMn2O5 phase [To better crystallize the structure and resolve the real crystal symmetry of the perovskite phase obtained by mechanochemistry, the PBMO-m sample was annealed for 3 h at increasing temperatures in an inert Ar atmosphere. O5 phase .2O5 phase can be obtained from the disordered Pr0.5Ba0.5MnO3 or the fully oxidized PrBaMn2O6 phases when they are annealed at 800–900 °C in a reducing atmosphere [2O6 and GdBaMn2O6 up to 980 °C under an inert atmosphere leads to the formation of the half-reduced phase with an oxygen stoichiometry close to 5.5, the same treatment for PrBaMn2O6 only produces a slight decrease in the oxygen content in the oxidized phase [0.5Ba0.5MnO3 was obtained by mechanochemistry, the reduced layered phase was formed when the PBMO-m sample was annealed (above 800 degrees) in an inert atmosphere.As mentioned above and according to the literature, the fully reduced PrBaMnmosphere ,18. Moreed phase . TherefoTo obtain new insights into the nature of the perovskite phase obtained by mechanochemistry and to estimate the oxygen content before and after the annealing treatments in Ar, thermogravimetric measurements were performed in different atmospheres. The main results are depicted in 0.5Ba0.5MnO3 to PrBaMn2O5 (~3.3 wt.%) and it is the consequence of the presence in the product of BaCO3, which decomposes in the temperature range studied. However, when the PBMO-m + 900 °C/Ar and PBMO-m + 1100 °C/Ar samples were heated in air, a weight gain was observed in the 200–300 °C temperature range  was obtained when PBMO-m was annealed in Ar; the use of a reducing atmosphere is not necessary.A weight loss of ~4.8% was observed when the PBMO-m sample was heated up to 850 °C in a flowing Ar atmosphere. This weight loss a is even+ 1100 °C/Ar + TG/air. The XRD pattern of this last sample was consistent with a tetragonal P4/mmm structure with lattice parameters a = 3.9004(1) Å and c = 7.7582(2) Å in agreement with the literature values for PrBaMn2O6 [In rBaMn2O6 ,26.A microstructural characterization was carried out for the PBMO-m, PBMO-m + 1100 °C/Ar and PBMO-m + 1100 °C/Ar + ATG/air samples. According to SEM micrographs obtained for the three samples , the morPm-3m (a = 3.8962(3) Å). The corresponding (h k l) planes are marked in the figure.A TEM and related techniques study was carried out to deep into the microstructure of the three samples. The results obtained for sample PBMO-m are depicted in P4/nmm; 129) and the tetragonal fully oxidized layered phase  is very slight. They only diverge in a 45 degree rotation at the base of the structure in such a way that a129= √2a123 and the c parameter is nearly the same. For this reason, it is quite difficult to differentiate both structures by SAED. In the case of the PBMO-m + 1100 °C/Ar sample, several high-resolution micrographs were found, where the FFTs could be indexed to both structures. This fact can be seen when comparing FFTs with the electron diffraction ED calculated diagrams for both symmetries (P4/nmm structure. A representative image is depicted in 129 zone axis and the (h k l) are marked. On the other hand, the microstructural analysis of the PBMO-m + 1100 °C/Ar + ATG/air sample 123 zone axes, respectively. The (h k l) planes are marked in the figure. Therefore, it can be confirmed that the reduced and oxidized layered samples possess P4/nmm and P4/mmm tetragonal symmetry, respectively, in good agreement with the found XRD results.mmetries c,d. Hower sample , the oxiThe chemical composition (regarding the cations) of the three samples  was analyzed by EDX spectroscopy and the corresponding spectra and average atomic percentage of the cations are presented in 2 and synthetic air from 100 to 800 °C. Remember that after the sintering process in Ar atmosphere, the reduced layered PrBaMn2O5+δ phase was obtained. The electrical conductivity reaches a maximum of 21.1 S·cm−1 when reduced and 96.8 S·cm−1 in its oxidized state (measurement in air atmosphere). The very high electrical conductivity values reported for this layered PBMO double perovskite, particularly in air, are comparable to the values obtained by Sendogan et al. [n et al. , indicat•= Mn•Mn). According to the Kröger–Vink equation, the interaction between oxygen vacancies and mobile electronic holes can be expressed as followsIt is known ,47 that 2+ to Mn3+ and even to Mn4+ ions . In contrast, in a reducing atmosphere, the electroneutrality is maintained by the formation of oxygen vacancies followed by the reduction of Mn4+ to Mn3+ to Mn2+ ions.This relationship implies that the concentration of oxygen vacancies will increase under a reducing atmosphere, yielding in enhanced oxide-ionic conductivity and oxygen ion transfer sites between the anode and the electrolyte. In an oxidizing atmosphere, the electrical charge is mainly balanced by the conversion of MnThe thermal dependence of conductivity for oxygen ionic conductors can be obtained from the well-known Arrhenius plot. The activation energy values of 0.06 and 0.33 eV were found in the oxidized and reduced specimens, respectively. The slopes obtained, and thus the activation energies calculated from the linear fits, are also consistent with data reported previously in layered PBMO double perovskites fabricated by other synthesis routes.6O11, BaO2 and MnO powder mixture with Pr/Ba/Mn stoichiometric ratios of 1/1/2 led to the formation of a pseudo-cubic perovskite phase consistent with Pr0.5Ba0.5MnO3. The presence of the hexagonal phase associated with the BaMnO3 secondary phase, as generally found in thermal-related synthesis processes, was not detected in this mechanochemical route, which yields in direct fabrication of the layered perovskite, without further annealing in an reducing atmosphere. If the perovskite phase obtained by mechanochemistry is annealed at an increasing temperature in an inert Ar atmosphere, the layered PrBaMn2O5+δ phase is formed. This phase transforms into the oxidized layered PrBaMn2O6-δ when treated in air at 200–300 °C. Electrical characterization shows very high electrical conductivity of layered PBMO, becoming therefore excellent candidates to be considered as SOFC electrodes.High-energy milling in air of a PrThe fabrication of a full SOFC containing a PBMO anode or a symmetrical fuel cell with PBMO in both electrodes, as well as the characterization of such fuel cell performance is planned for future experiments. In addition, considering the benefits of this fabrication method and the good properties of the fabricated PBMO double perovskite, authors plan to incorporate other elements  using this fabrication method in the double perovskite in order to further enhance the electrical properties.


Here, we review various ways ILCs are currently identified, focusing on potential problems and their solutions. The first step to identify all ILCs is to exclude other lymphocytes and myeloid cells by their lineage-specific markers (Lin). However, the Lin cocktail varies in various studies, and the definition of Lin- population containing ILCs is often ambiguous, resulting in contamination of LinWe have designed combinations of cell surface markers to identify ILC populations in various tissues of B6 mice by flow cytometry. To minimize T cell contamination, TCR/CD3ϵ antibodies were used separately from the Lin cocktail. ILCs identified by surface markers are confirmed by the expression of the transcription factors GATA3, RORγt, T-bet and Eomes.+NK1.1+TCR/CD3ϵ−CD49a+CD49b−. However, defining ILC1s in other tissues remains a challenge. ILC2s in the lung are identified by Lin−TCR/CD3ϵ− Thy1+CD127+ST2+ whereas ILC2s in the small intestine and liver are identified by Lin−TCR/CD3ϵ−Thy1+GATA3+RORγt−. ILC3s in B6 mouse spleen, liver, lung and small intestine are identified by Lin−TCR/CD3ϵ− Thy1+CD127+RORγt+.ILC1s in the B6 mouse liver are identified by Lin-NKp46The ILC population is heterogeneous and the strategies to identify ILCs have to be designed for each ILC population and tissue. Excluding T cells in all cases is crucial, and a combination of transcription factors GATA3, RORγt, T-bet, and Eomes should be used to identify ILCs. Using CD3ϵ/TCRs in a different fluorochrome not in Lin cocktail minimizes contamination of T cells specifically identify individual ILC populations in various tissues. For mouse ILCs, the Lin cocktail typically includes Ter119 for erythroid cells, Gr-1 for granulocytes, CD11c for dendritic cells, CD11b for monocytes/macrophages, CD19 for B cells, and CD3ϵ/TCR for T cells  consists of three groups, namely, ILC1, ILC2, and ILC3  can be identified by combinations of cell surface markers and transcription factors without contamination of other lymphocytes.22.1Rorc(γt)-eGFP reporter mice (JAX stock #007572) and Rag1−/− mice (JAX stock #002216) on a B6 background were originally purchased from Jackson Laboratory. Homozygous Rorc(γt)-eGFP mice were crossed with B6 mice to generate heterozygous Rorc(γt)-eGFP mice. All mice were maintained in the British Columbia Cancer Research Centre animal facility under specific pathogen-free conditions. The use of these mice was approved by the animal committee of the University of British Columbia and in accordance with the guidelines of the Canadian Council on Animal Care.C57BL/6J (B6) mice were bred in-house and purchased from breeders of Jackson Laboratory . 2.22 until respiratory arrest. Tissues were collected in a tube containing 5 ml of Dulbecco’s modified Eagle medium (DMEM) with 2% fetal bovine serum (FBS).All tissues including the liver, lung, spleen, and SI were processed as described previously in detail . Mice we2.2.1g) for hepatocyte sedimentation, the supernatant was collected and centrifuged . The pellet was resuspended in 16 ml of 40% Percoll. After centrifugation (room temperature (RT), 20 min, 1,400 × g), the pellet resuspended in 3 ml of red blood cell (RBC) lysis buffer .Livers were mashed and passed through 40-µm cell strainers  with 10 ml of DMEM  + 10% (v/v) FBS, and the strainers were washed with 15 ml of the same media. After centrifugation , the supernatant was discarded, and RBCs were lysed using RBC lysis buffer.Spleens were mashed through 70-µm strainers in 5 ml phosphate-buffered saline (PBS) + 2% FBS. Strainers were washed with 5 ml of the same buffer. After centrifugation , the supernatant was discarded, and the pellet was resuspended in 5 ml of 36% Percoll and centrifuged at RT, 20 min, 650 × g. RBCs were lysed using RBC lysis buffer.Lungs were minced in a 10-cm petri dish by using a razor and then placed in a tube containing 5 ml of digestion buffer . Tubes were incubated for 25 min at 37°C in a shaker at 200 rpm. Digested tissues were mashed and passed through 70-µm cell strainers by adding 5 ml DMEM + 10% FBS. After centrifugation  HBSS + 15 mM HEPES/pH = 7.2) and were cut into ~1-cm pieces after discarding the fat and Peyer’s patches. With the use of surgical scissors, each fragment was cut open longitudinally. Then, the luminal contents were rinsed three times using an intestine wash buffer. The intestinal epithelial cells were removed by incubation in CMF/EDTA/FBS buffer (HBSS + 15 mM HEPES + 10% FBS + 2.5 mM EDTA + 1 mM DTT/pH = 7.2) for 20 min at 37°C in a shaker at 250 rpm. The tubes were shaken manually 10 times, and then the contents were passed through sieves and rinsed with wash buffer. This step could be repeated for two more times. Tissues were digested using 100 U/ml of Collagenase VIII  and 9.41 U/ml of DNase I from bovine pancreas . Digestion buffer was prepared in DMEM containing 10% FBS. Tissue digestion was performed in a shaker for 25 min at 37°C and 5% CO2. The contents were filtered through a 100-μm cell strainer, and 10 ml of intestine washing buffer was added. After centrifugation , the pellet was resuspended in 5 ml of PBS containing 2% FBS and filtered through 70-μm and then 40-μm cell strainers. Cells were centrifuged , and then RBCs were lysed using RBC lysis buffer.The SIs were extensively washed with intestine wash buffer (Cag). The supernatant was discarded, and the pellet was resuspended in 1 ml PBS + 2% FBS and then transferred to a 5-ml flow tube through a 35-μm strainer cap. Cells were then stained with antibodies in PBS containing 2% FBS.After adding RBC lysis buffer, cells were incubated for 5 min at RT. DMEM at a volume of 5 ml with 2% FBS was added and centrifuged  1640 media containing 10% FBS, 100 U/ml of P/S, 50 mM of 2-ME, Brefeldin A , 30 ng/ml of phorbol 12-myristate 13-acetate , and 500 ng/ml of ionomycin . Intracellular cytokine staining was performed after the incubation and surface staining using Cytofix/Cytoperm Fixation/Permeabilization Solution kit (BD Biosciences) according to the manufacturer’s protocol. Intracellular GATA3, RORγt, T-bet, and Eomes staining were carried out similarly without preincubation using Foxp3/Transcription Factor Staining Buffer Set (Thermo Fisher Scientific) according to the manufacturer’s protocol. Simultaneous intracellular RORγt and cytokine staining were performed using Foxp3/Transcription Factor Staining Buffer Set according to the manufacturer’s protocol.2.4Single-cell suspensions were incubated with anti-mouse CD16/32 antibody (clone 2.4G2) to block non-specific binding to Fc receptors before surface staining. Antibodies used in this study are listed in 33.1To accurately identify ILC1s in the liver under steady-state conditions, we performed 12-color flow cytometry Table 2 −NK1.1+NKp46+CD3ϵ/TCRs− cells can be divided into CD49b+CD49a− NK cells and CD49b−CD49a+ILC1s. The former express Eomes and T-bet, whereas the latter express Eomes−T-bet+  (−Thy1+CD3ϵ/TCRs−ST2+GATA3+) also express CD25  or allophycocyanin (APC). ILC2s are then gated by Lin−hy1+CD3ϵ/TCRs−GATA3+RORγt−) are ST2−, but approximately 90% express KLRG1, and over 90% are IL-17RB positive (−Thy1+CD3ϵ/TCRs−GATA3+RORγt−) can be divided into ST2+ (approximately 70%) and ST2− populations. Approximately 50% of them are KLRG1+, and over 90% are IL-17RB+ -eGFP reporter mice. ILC3s in these mice can be detected by Lin−Thy1+NK1.1−CD3ϵ/TCR−CD127+GFP+. ILC3s are mostly IL-18R+ and CD49a+ , and the Lining ILCs , 31. Linytometer .−NKp46+NK1.1+CD49a+CD49b−. The combination of CD49a and CD49b clearly separates ILC1s from NK cells, whereas NK1.1 expression excludes ILC3s. This can be confirmed by the expression of the NK-associated transcription factor Eomes in CD49a−CD49b+, while the CD49a+CD49b− population lacks Eomes and the ILC3-associated transcription factor RORγt. It should be noted that CD49a is expressed not only on ILC1s but also on other lymphocytes, including ILC3s and NKT cells, and it is important to have CD3ϵ/TCR in separate fluorochrome to avoid contamination of T cells as discussed above. This strategy cannot be universally applied to ILC1s in all tissues and other strains of mice lacking NK1.1. It has been reported that salivary gland ILC1s express CD49b and Eomes, and intestinal ILC1s also express Eomes  can be used in place of ST2 to identify ILC2s. However, recently reported IL-18R IL-17RB , and IL-+CD127− and ST2−CD127− cells in the lung of Alternaria-treated mice -eGFP reporter mice (Rorc(γt)-eGFP mice are RORγt-deficient, only heterozygous mice can be analyzed for ILC3s. Due to a half-dose of RORγt in the heterozygous mice, ILC3 numbers in the heterozygous mice are lower than wild type. Because RORγt is also expressed in some T cells, T cells have to be excluded from ILC3 analyses by the strategy described above for ILC1s and ILC2s. A subpopulation of ILC3s also expresses NKp46 but not NK1.1, while NK cells and ILC1s express both. In our study, NK1.1 was used to discriminate ILC3s from ILC1s and NK cells.ILC3s share many surface markers with other ILCs; to identify and distinguish ILC3s from other ILCs, analysis for RORγt expression is critical. RORγt expression also distinguishes ILC3s from the IL-18Rter mice . The latIn summary, the strategies to identify ILCs have to be designed for each ILC population and tissue due to the heterogeneity of ILCs. In all cases, it is critical to exclude T cells, and the identity of ILCs should be confirmed by a combination of transcription factors GATA3, RORγt, T-bet, and Eomes.The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.The use of these mice was approved by the animal committee of the University of British Columbia and in accordance with the guidelines of the Canadian Council on Animal Care. The study was conducted in accordance with the local legislation and institutional requirements.MS, DK, and MO wrote and edited the manuscript. MS, DK, and MO performed the experiments and generated figures and tables. FT reviewed the drafts, provided critical input, and edited the manuscript and figures. All authors contributed to the article and approved the submitted version.