Saccharomyces cerevisiae, the transcriptional repressor Opi1 regulates the expression of genes involved in phospholipid synthesis responding to the abundance of the phospholipid precursor phosphatidic acid at the endoplasmic reticulum. We report here the identification of the conserved leucine zipper (LZ) domain of Opi1 as a hot spot for gain of function mutations and the characterization of the strongest variant identified, Opi1N150D. LZ modeling posits asparagine 150 embedded on the hydrophobic surface of the zipper and specifying dynamic parallel homodimerization by allowing electrostatic bonding across the hydrophobic dimerization interface. Opi1 variants carrying any of the other three ionic residues at amino acid 150 were also repressing. Genetic analyses showed that Opi1N150D variant is dominant, and its phenotype is attenuated when loss of function mutations identified in the other two conserved domains are present in cis. We build on the notion that membrane binding facilitates LZ dimerization to antagonize an intramolecular interaction of the zipper necessary for repression. Dissecting Opi1 protein in three polypeptides containing each conserved region, we performed in vitro analyses to explore interdomain interactions. An Opi11-190 probe interacted with Opi1291-404, the C terminus that bears the activator interacting domain (AID). LZ or AID loss of function mutations attenuated the interaction of the probes but was unaffected by the N150D mutation. We propose a model for Opi1 signal transduction whereby synergy between membrane-binding events and LZ dimerization antagonizes intramolecular LZ–AID interaction and transcriptional repression.In  Saccharomyces cerevisiae is the founding member of a transcriptional repressor protein family defined by the array of three domains: the leucine zipper (LZ), the uncharacterized conserved region (UCR), and the activator interacting domain (AID)  synthesis, and lipid homeostasis (B)  phenotype, where inositol biosynthesis exceeds cellular needs. Ino2 and Ino4 belong to the basic helix-loop-helix family of transcriptional activators, and as a heterodimer, they drive transcription upon binding to cis-regulatory elements at the promoter of targeted genes. Opi1 is the transducer of the regulon. Opi1 interacts through its AID with Ino2 preventing transcription, with mutants bearing truncations of this domain or point mutations affecting conserved residues defective in Opi1-repressing function (Opi1 from in (AID) A and S1.albicans , 4, 5, 6eostasis B  and to the integral ER protein Scs2 , 14, 15.Figs. 1ormation , 23, 24,ormation , 27, 28.henotype , 29.OPI1 alleles and screened for those that were resistant to derepressing conditions and identified the LZ as a hot spot for gain of function mutations. The characterization of the variant Opi1N150D reported here, affecting a canonical asparagine residue of the LZ, implies that LZ-mediated homodimerization attenuates Opi1 repression. We propose a model whereby membrane binding through the PA-binding region and FFAT motifs facilitates LZ dimerization, antagonizing the intramolecular interaction of the LZ to the AID, required for Opi1 transcriptional repression of lipid-synthesizing genes.In this study, we sought to further understand the mechanisms by which Opi1 transduces membrane signals into gene expression regulation. We generated a collection of S. cerevisiae, phosphatidylcholine (PC) is the end-product for PL synthesis through both CDP-diacylglycerol and CDP-choline branches, working as a sink for phosphatidylserine and phosphatidylethanolamine metabolism . Interestingly, all the Opi1 mutants derived from this screen were in the LZ domain.Aiming to identify structural elements of Opi1 responding to Nte1-mediated lipid metabolic changes, we performed a genetic screen for Opi1 allelic variants that were refractory to Nte1-increased expression. Considering the direct role of Scs2 and its paralog Scs22 towards Opi1 membrane association, we selected a henotype A and 2A.scs2Δ scs22Δ cells expressing Opi1WT grew at 37 °C when carrying the pRS416-NTE1 plasmid, cells expressing any of the mutated variants performed poorly under the same condition. However, these variants imposed a growth limitation for scs2Δ scs22Δ cells independently of NTE1 dose when growth was assessed at 30 °C (A). We re-assessed the susceptibility of these variants to increased Nte1 expression in the presence of Scs2/Scs22. The OPI1 alleles were expressed in an opi1Δ strain carrying or not NTE1 gene on a plasmid and their growth phenotype examined (B). Whereas the increased expression of Nte1 alleviated the ino phenotype elicited by Opi1 variants carrying the mutations L143S, H144L, H144Y, or K147E, it was unable to reverse the growth impairment upon the expression of Opi1N150D.Further characterization of each of the Opi1 LZ variants isolated was undertaken. Whereas at 30 °C A. We re-examined B. WhereaC142Y was isolated through a genetic screen  mutations. Opi1c screen  and Opi1e zipper   . We assessed the phenotype arising from the N150I mutation. Isoleucine is the most frequent residue found at a positions of canonical zippers. Contrarily to the charged variants, Opi1N150I conferred a growth phenotype very similar to the WT allele across conditions of increasing inositol demand  , 43, 44.tion (A) . Importahe dimer , 46, 47.he dimer , 46, 47.mitation C. We assl demand , confirmmitation , suggestOPI1 from two plasmids in an opi1Δ strain and assaying growth under inositol limitation. Three previously known LOF mutations identified in the UCR: K226E, L252F, and L255S and two identified in the AID: S339F and V343E . The phenotype imparted by Opi1N150D was not prevented by the co-expression in trans of any of these variants. However, it was abrogated or attenuated when these mutations were present in cis of the N150D mutation, supporting the notion that a variant unable to dimerize is dominant but implying that mere monomerization does not confer repression. The functional integrity of either the UCR or AID-conserved domains is necessary for Opi1 to acquire repression.If LZ dimerization antagonizes monomerization and repression, an Opi1 variant unable to dimerize would be dominant. We explored this notion by co-expressing different alleles of nd V343E   was similar to Opi1WT-mGFP. Confocal microscopy examination showed that all the variants were nucleoplasmic for inositol-fed cells (A). After 2 h of inositol withdrawal, Opi1WT-mGFP acquired its typical perinuclear and cortical ER localization whereas Opi1N150D-mGFP as well as the LOF variants remained nucleoplasmic. For the LOF variants, it is conceivable that upon the onset of the opi phenotype by derepressed INO1 transcription, the consequent increase of inositol and PI biosyntheses leads to a reduction of PA pools, Opi1 membrane detachment, and nucleoplasmic localization. Aiming to dissociate Opi1 localization from the phenotype of the mutants, we re-assessed the LZ variants in haploids cells carrying a KO allele of the INO1 gene. Growth plate assays showed that ino1Δ cells performed like WT cells provided 5 μM inositol was present in the media . When ino1Δ cells were shifted into 5 μM inositol-containing media, Opi1WT-mGFP acquired perinuclear distribution after 6 h of cultivation whereas Opi1N150D-mGFP remained nucleoplasmic. In contrast with the observation for the INO1 parental strains, the three LOF variants acquired perinuclear localization when ino1Δ cells were grown on low inositol media for at least 6 h. The relocalization of the LOF LZ alleles indicates that the functionality of the LZ is not essential for Opi1 membrane association but necessary for repression upon membrane detachment. The persistent nucleoplasmic distribution of Opi1N150D-mGFP supports the notion that the inability to dimerize by this variant locks Opi1 in a detached and repressive form.To gain insight about the role of the LZ into Opi1 localization and function, Opi1 was tagged at its C terminus with monomeric-GFP. Both Opi1terparts . We expll allele . For Opin the LZ . The expgulation . This obed cells A. After he media . As obseng media B. When iC142Y, Opi1L160A, and Opi13L surpassed the hemizygous WT and performed like the null allele under inositol limitation and upon further stress by the addition of tunicamycin  (A and C) carrying the WT or the N150D variant (A and B). The elution profiles for MBP-N-Opi1WTversus MBP-N-Opi1N150D were distinct. Both affinity preparations rendered high molecular weight aggregates eluting at the void volume of the column. The earlier elution of MBP-N-Opi1WT (peak I) compared to MBP-N-Opi1N150D (peak II) implies a larger complex with estimated molecular weights of 190 kDa for peak I versus 100 kDa for peak II. Electrophoretic analyses of the eluates from the SEC showed that a polypeptide of the expected molecular weight of 65 kDa for the MBP-N-Opi1WT and MBP-N-Opi1N150D polypeptides was the main constituent of both peaks (B). The chromatographic analyses are consistent with MBP-N-Opi1 forming a higher order structure, likely a dimer via the LZ, and support the notion that an ionic residue at the LZ position a 150 of Opi1 prevents dimerization.To directly examine the role of the N150D amino acid change within the LZ dimerization interface on the capacity of Opi1 to dimerize, we analyzed by size-exclusion chromatography (SEC) affinity purified preparation of maltose-binding protein (MBP) fused to the N-terminal Opi1 -N-Opi1) , A and C variant , A and Bth peaks B. The chA), it is conceivable that changes within the LZ of Opi1 might affect binding to PA. We assessed binding for SEC-purified MBP-N-Opi1WT and MBP-N-Opi1N150D to PA-containing liposomes. MBP-N-Opi1N150D binding to PA-containing liposomes was comparable to MBP-N-Opi1WT, whereas the binding to PC-only liposomes was negligible for both (C). This observation suggests that the strong phenotype elicited by Opi1N150D variant is not due to a severe defect in PA binding and is consistent with the observation that the LOF LZ variants are sensitive to inositol-driven membrane changes.As the LZ is C-terminally adjacent to the PA-binding motif of Opi1 A, it is for both C. This oin vitro pull-down assays using recombinant tagged peptides. The Opi1 polypeptide was dissected into three regions containing each conserved domain (A): N-Opi11-190, M-Opi1191-290, and C-Opi1291-404. They were subcloned C-terminally of MBP and of glutathione-S-transferase (GST), and the recombinant chimeras were expressed in bacteria and affinity purified (B). Pull-down assays were conducted as follows: the prey was presented to the bait carrying the alternate tag and bound to its affinity resin. After washing the beads and upon specific elution of the bait from its resin, the presence of the prey in the eluate was assessed by Western blotting. The N-Opi1 fusion proteins interacted with each other consistent with the LZ-driving Opi1 dimerization. In addition to self-association, the N-Opi1 chimeras interacted with C-Opi1 chimeras (A). The chimeras MBP and GST-M-Opi1 carrying the UCR did not interact with themselves or with N-Opi1 or C-Opi1. Thus, regardless of the tag and regardless of their presence as a bait or as a prey, the known self-association of N-Opi1 was observed in all combinations. Interestingly, we also observed N-Opi1 interacting with C-Opi1 under all conditions.To explore Opi1 domain interactions, we performed d domain A: N-Opi1purified B. Pull-dchimeras A. The chC) and their binding towards GST or MBP-C-Opi1WT, respectively, were assessed by pull-down assays. To challenge the specificity of these interactions, binding reactions were carried out at decreasing reactant concentrations. Irrespective of the configuration of the pull-down assay, the 3L mutation attenuated the interaction whereas the binding of N-Opi1N150D to C-Opi1WT was comparable to N-Opi1WT (B). We also assessed the impact of loss of function mutations S339F and V343E (D) on the interaction of C-Opi1 to N-Opi1WT by pull-down assays. The fraction of GST-C-Opi1S339F and of GST-C-Opi1V343E that remained bound to MBP-N-Opi1WT was reduced compared to GST-C-Opi1WT (C). Altogether, the in vitro pull-down data is consistent with the notion that beyond Opi1 LZ self-association, a second LZ interaction with the Opi1 AID occurs.We next examined if N150D and 3L mutations affected the interaction between N-Opi1 and C-Opi1. These LZ variants were expressed as MBP or GST-N-Opi1 fusions, affinity purified C and theN-Opi1WT B. We alsnd V343E D on the C-Opi1WT C. AltogeL143S, Opi1H144L, Opi1H144Y, and Opi1K147E variants to Nte1-mediated rescue depended on Scs2/Scs22   . Howevernte1Δ and gpc1Δ cells exhibited a less saturated PC profile under those conditions (nte1Δ cells with both having a MUPC/DUPC ratio of 0.3 (A). Under inositol starvation, the PC from WT cells exhibited a more saturated fatty acid profile, whereas in nte1Δ cells, there was a less saturated PC profile resulting in a reduction of the MUPC/DUPC ratio from 1.1 to 0.8, confirming a previous observation . Overall, the data are consistent with Nte1 preferentially hydrolyzing DUPC species. One prediction from this data would be Nte1 activity increases the unsaturated fraction of the cellular pool of FFA.Aiming to gain more insight into the ability of Nte1 to overcome Opi1 repression, we examined its contribution towards PC homeostasis. Importantly, inositol availability is known to (i) impact the PC fatty acyl species distribution profile and (ii) drive flux through the Kennedy pathway for PC synthesis and its turnover by Nte1 , 48, 49.o of 0.3 A. Under ol cells B. OveralA and B). The absence of Nte1 led to a significant decrease on the expression of the reporter when inositol-starved cells were examined, whereas increasing NTE1 gene dosage elevated luciferase expression for inositol-fed cells (B and C). The differential activation of this reporter upon Nte1 variation reveals the contribution of Nte1 to the onset of this response and is consistent with Nte1 preferentially increasing the cellular level of free OA and simultaneously decreasing the unsaturated fatty acid content of PC.To explore if unsaturated FFA level in the cell is modulated by inositol availability and if Nte1 contributes to this regulation, we took advantage of the oleic acid -activated transcription factor Oaf1. Oaf1 directly binds OA and regulates the expression of genes involved in fatty acid β-oxidation , 51, 52.ed cells , B and Cd positions of the heptads  and L143, I157, and V164 at the a positions (A). The asparagine at the reminder a 150 position is thought to dictate specificity by allowing electrostatic bonding across of the otherwise hydrophobic dimerization interface and to modulate affinity by reducing the stability of the dimer. Instead, an ionic residue would prevent LZ homodimerization by charge repulsion. It was shown that the recombinant chimera MBP-Opi11-180 undergoes dimerization in a concentration-dependent manner, suggesting the LZ allows dynamic dimerization  medium or synthetic complete medium . Medium was supplemented alternatively as required for plasmid maintenance with SC drop-out mixes. Of note, inositol concentration for SC media is 486 μM, yeast nitrogen base contributing 11 μM, and SC mix contributing 475 μM. Yeast cells were routinely grown at 30 °C in synthetic defined medium (0.67% yeast nitrogen base without inositol and without amino acids) supplemented as required for plasmid maintenance and nutritional auxotrophies and with the indicated amount of inositol.OPI1 alleles, a haploid opi1Δ BY 4742 was generated by sporulation and selection of a heterozygous diploid OPI1/opi1Δ::KanMX4 BY4743 obtained from Euroscarf. The screen strain BY4742 opi1Δ::HYG scs2Δ::NAT scs22Δ::KanMX4 was generated by standard genetic crosses of single mutants obtained from a BY4741 deletion collection. The genetic KanMX4 marker was swapped into nourseothricin acetyltransferase or hygromycin resistance markers using plasmid pAG25 or pAG32 as described (For the characterization of escribed . Other sOPI1 ORF cloned contiguous and jointed through an XmaI site. A DNA fragment extending from nt −274 to +3 of OPI1 gene (numbered from start codon) was amplified with primers 5′-U-OPI1-SalI and 5′-D-OPI1-XmaI and positionally cloned into pRS415 vector using those restriction enzymes. A second DNA fragment from nt 1209 to 1597 of the OPI1 gene including OPI1 stop codon (nt 1213–15) was generated by PCR using primers 3′-U-OPI1-XmaI and 3′-D-OPI1-SacI and subcloned contiguous of the 5′ end of OPI1 into the pRS415 backbone using XmaI and SacI to generate pPM170.Plasmid pPM170 carries both upstream and downstream regions of XmaI/NsiI double digestion for subcloning the mutagenized OPI1 PCR amplicon by in vivo plasmid gap repair. At both ends of the mutagenized OPI1 fragments, there is ∼0.1 Kb overlapping with the 5′ and 3′ flanking regions of OPI1 gene present in the pPM170 plasmid. The WT allele of the OPI1 gene was cloned by PCR amplification from genomic DNA using primers 5′-U-OPI1-SalI and 3′-D-OPI1-SacI and positional subcloned into vector pPM170 by inserting a 1.7 Kb NdeI/XbaI fragment to render plasmid pPM169. Primers used in this work are listed in The pPM170 plasmid was linearized by OPI1 alleles encoding the variants C142Y, L143S, H144Y, K147E, and L160A were subcloned from their original plasmid source into plasmid pPM169 by replacing the 0.6 Kb AatII/BglII DNA fragment. The allelic variants H144L and N150D were introduced by site-directed mutagenesis into pPM169. The variants L153S L160A and L146A L153S L160A (3L) were constructed by sequential site-directed mutagenesis of the allele encoding the L160A variant carried on a pRS415 plasmid. The alleles encoding the variants K226E, L255S, S339F, and V343E were cloned from genomic DNA by PCR amplification followed by swapping a 0.45 Kb fragment generated by BglII and SnaBI double restriction digestion into pPM169 backbone. The allelic variant L252F was subcloned from its original plasmid source by BglII/SnaBI 0.45 Kb fragment swapping. All the cis constructions carrying the LOF mutations located downstream of the BglII site were built by 0.45 Kb BglII/SnaBI fragment swapping into a pRS415-OPI1N150D backbone. For the construction of the double N150D K226E mutant, the N150D mutation was introduced by site-directed mutagenesis into the TOPO clone carrying the mutation K226E for subsequent subcloning of the 0.6 Kb AatII/BglII DNA fragment into pPM169. Plasmids used in this study are listed in The Taq polymerase reaction buffer, 5 pmol of 5′-OPI1-M primer, 5 pmol 3′-OPI1-M primer, and 2 units of Taq DNA polymerase. Mutagenic conditions were achieved by varying deoxynucleotide concentrations  and increasing divalent cation concentration (4.5 mM MgCl2 and 0.5 mM MnCl2). Several pools of amplicons generated by error-prone amplification of OPI1 gene were subcloned by gap repair into linearized pPM170. Briefly, the gapped plasmid plus the PCR-amplified OPI1 products were cotransformed into an opi1Δ scs2Δ scs22Δ carrying pRS416-NTE1 plasmid. For gap repair, 120 ng of PCR product was cotransformed with 60 ng of XmaI/NsiI double-digested pPM170 plasmid into 2.5 × 107 cells. Transformants were selected in SC media minus uracil and leucine. Typically, 4000∼6000 colonies were obtained from a standard transformation reaction. Colonies were replicated onto synthetic define selective media lacking inositol at 37 °C and those performing poorly under those conditions were further analyzed.Mutagenic PCR reactions were performed in 25 μl containing 10 ng of yeast genomic DNA, 1x OPI1 alleles, a 1.3 Kb DNA fragment containing the 3′ end of OPI1 ORF fused to GFP-encoding sequence was amplified from genomic DNA of a strain encoding OPI1-GFP::HIS3 with primers F2-G and 3′-GFP-HIS3-SacI. The mutation A206K preventing GFP dimerization was introduced by site-directed mutagenesis. This fragment was subcloned downstream of the OPI1 alleles carried on a pRS415 vector by replacing the SnaBI/SacI fragment to render Opi1-mGFP ORF under the control of OPI1 promoter. A 0.4 Kb DNA fragment containing the 3′ region downstream of OPI1 ORF was PCR amplified from genomic DNA using primers AscI-3′-Up-OPI1 and 3′-D-OPI1-SacI. This fragment was subcloned downstream of OPI1-mGFP ORF, carried on a pRS415 backbone, by restriction digestion and ligation at the AscI and SacI sites. The resulting plasmids carrying OPI1-mGFP variants flanked by the upstream and downstream sequences of OPI1 gene served as donors of healing DNA fragments for a double strand break introduced by Cas9 at the kanMX4 gene of an opi1Δ::kanMX4 haploid strain. The healing fragment was generated by XhoI digestion and isolation of the 2.5 Kb DNA fragment. Haploid opi1Δ::kanMX4 cells were transformed with the healing DNA fragment and with plasmid pAR1275. This pRS425-based plasmid expresses Cas9 and encodes a guide RNA that targets kanMX4 for a double strand break. Transformed cells were selected on SC-LEU plates; isolated colonies were propagated under nonselecting conditions for plasmid lost and were assessed for G-418 sensitivity as it was regained upon recombination at the OPI1 locus. Proper integration of the healing fragment was assessed by PCR using primers 5′-OPI1g and 3′-OPI1g. Positives clones were characterized phenotypically.To generate the Opi1-mGFP fusions for the studied g for 5 min. A 3 μl aliquot was mounted on thin 0.9% Sea Kem agarose pad prepared in synthetic define medium containing the indicated inositol concentrations. Images of live cells were captured using a Leica TCS SP8 with LIGHTNING confocal microscope using excitation wavelength of 448 nm at 100x magnification. A 1.7× magnification was further applied to all images by the LAS X software (https://www.leica-microsystems.com/products/microscope-software/p/leica-las-x-ls/). Fields were focused manually using DIC. Exposure times and laser intensities were kept constants for all different yeast strains.Log phase cells grown in the indicated media were concentrated by centrifugation at 10007 logarithmic cells were immersed in an ice-water bath and media was made to 10 mM NaN3, 10 mM NaF, and 1 mM PMSF. Cultures rested at 4 °C for 5 to 10 min, cells were centrifuged, resuspended in ice-cold 10 mM NaN3, 10 mM NaF, and 1 mM PMSF, total number of cells equalized across samples and transferred into a screw-cap plastic vial, centrifuged again, and the cell pellet frozen using liquid nitrogen and stored at −80 °C. Cells were thawed in 250 μl of ice-cold 1X Laemmli sample buffer containing 5% β-mercaptoethanol and 1 mM PMSF, and cold glass beads were added up to the suspension meniscus. Cells were lysed by four cycles of 30 s of bead beating intercalating with 60 s on ice. Cell lysates were recovered and clarified by centrifugation at 21,000g for 10 min at 4 °C. Appropriated aliquots were analyzed by SDS-PAGE.Flasks containing cultures of around 5 × 10nm determined, and ten-fold serially diluted from an initial A660nm of 0.4 in sterile water. Cell suspensions were pinned down onto solid media and incubated for 2 to 4 days at the indicated temperature.Singles colonies were grown overnight at 30 °C in 1.5 ml of the indicated media. Cells were washed twice with ice-cold water, their OD at 660A) were amplified by PCR with the primers listed in OPI1 DNA templates as indicated. Amplified DNA was subcloned into the pCR 2.1-TOPO vector, DNA inserts were sequenced to ensure fidelity, and subcloned into the pGEX-4T-1 or pMAL-c expression vectors using the restriction enzymes indicated for each pair of primers. Recombinant proteins were expressed in BL21-derived NEB Express cells. Bacterial cells from an overnight culture were centrifuged and reinoculated at A660nm ∼0.15 in fresh LB media. For cells carrying pMAL vectors, LB media was supplemented with 0.2% dextrose. Cells were cultured at 37 °C until an A660 nm ∼0.7 was reached and recombinant protein expression was induced at 37 °C for additional 1.5 h by the addition of 1 mM IPTG. Cultures were chilled on an ice-water bath, pelleted at 4 °C, washed once with ice-cold 20 mM Tris–HCl pH 8, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, and 1 mM DTT, frozen using liquid nitrogen, and stored at −80 °C.DNA encoding the N-terminus of Opi1 from aa1 to aa190 , the middle fragment from aa191 to aa290 , and the C terminus from aa291 to aa404  A were amg and supernatants were transferred into new tubes. The supernatants were made 20% glycerol 0.05% Triton X-100 and added to 400 μl of amylose resin or glutathione-Sepharose respectively, previously equilibrated in binding buffer . The binding proceeded for 1 h at 4 °C on a spinning wheel, and the beads were washed 4 times with 10 ml of ice-cold binding buffer. Half of the washed slurry was kept at 4 °C and the other half was subjected to elution using 10 mM maltose or 10 mM glutathione in binding buffer, accordingly. Aliquots of the eluates were analyzed by SDS-PAGE followed by Coomassie blue staining to estimate recombinant protein enrichment and concentration . Statistical analyses were carried out using Prism software (https://www.graphpad.com/features).All primary antibodies used for Western blotting were of mouse monoclonal origin. Antibodies against yeast Pgk1  and against GST  were from Thermo Fisher Scientific. Antibody against MBP was from New England Biolabs . Antibody against GFP was from Takara . Antibody against firefly luciferase was from Abcam . Routinely, Western blotting images based on mouse monoclonal antibodies recognition were acquired using secondary goat anti-mouse IRDye-antibodies from LiCor Biosciences  by scanning on an Odyssey v3.0 (LiCor) and quantified with Fiji ImageJ (NIH) . Binding reaction proceeded at room temperature for 45 min in 150 μl of 20 mM Tris–HCl pH 7.4, 150 mM NaCl containing 1.6 mM liposomes and 0.5 μM of the indicated SEC-purified MBP-peptide probe. At the end of the incubation period, the bound protein fraction was separated by liposome flotation on discontinuous sucrose gradients. Succinctly, 100 μl of 75% sucrose prepared in 20 mM Tris–HCl pH 7.4, 150 mM NaCl was gently mixed with the reaction mixture to render 250 μl of 30% sucrose followed by the successive and careful addition of a layer of 200 μl of 20% sucrose and 50 μl of 0% sucrose in the same buffer. Centrifugation proceeded at 22 °C for 1 h at 85,000 rpm in a TL-100 ultracentrifuge using a TLA 120.1 rotor (Beckman Coulter). Four fractions, two of 100 μl and two of 150 μl, were collected from top to bottom and appropriate aliquots were analyzed by SDS-PAGE, stained with Instant Blue, and thoroughly destained with water. Images were acquired using Odyssey v3.0 (LiCor) and quantified with ImageJ (NIH). Bound fraction represents the abundance of the probe in the top fifth fraction relative to its total amount, distributed along the sucrose gradient. Statistical analyses were performed using Prism software. For the preparation of the affinity-purified protein probes, a frozen bacterial pellet from 1L culture was resuspended in 50 ml 20 mM Tris–HCl pH 7.4, 150 mM NaCl supplemented to 1 mM PMSF, 3 mM DTT, 30 U/ml Benzonase nuclease, 2 μg/ml pepstatin A, and 1× Complete protease inhibitor cocktail. Cell suspensions were sonicated four times for 30 s with 1 min resting in an ice-water bath each cycle. Crude cell extracts were centrifuged at 4 °C for 30 min at 46,000g. Supernatants were transferred into new screw-cup tubes containing 1 ml of amylose-resin equilibrated in 20 mM Tris–HCl pH 7.4, 150 mM NaCl, and affinity binding proceeded for 1 h at 4 °C with gentle rotation. Amylose beads were washed twice in batch with ice-cold 20 mM Tris–HCl pH 7.4, 150 mM NaCl and transferred into a chromatography column, followed with further washes until protein content neared baseline. MBP fusions were eluted by the addition of 10 mM maltose in 20 mM Tris–HCl pH 7.4, 150 mM NaCl. Fractions of 0.5 ml were collected and those containing the highest MBP-probe enrichment were pooled. MBP-probes content was assessed by SDS/PAGE and its relative abundance estimated. Around 0.5 mg of affinity purified MBP-N-Opi1WT and MBP-N-Opi1N150D were subjected to SEC on a Superdex 200 increase 10/300 Gl column, equilibrated, and developed with 20 mM Tris–HCl pH 7.4, 150 mM NaCl at 0.5 ml/min using an AKTA 25 platform. Fractions of 250 μl were collected after 0.2 column volume was eluted. Molecular weights were estimated based on the calibration of the SEC column using the following markers: Blue-Dextran (Vo), thyroglobulin (670 kDa), β-amylase (200 kDa), alcohol dehydrogenase (150 kDa), bovine serum albumin (67 kDa), carbonic anhydrase (29 kDa), and cytochrome C (12.5 kDa).Binding activity to PL membranes for the MBP-N-Opi12O, and flash frozen in liquid nitrogen. To 5 OD units of cells, 0.5 ml of CHCl3:CH3OH (1:1) and 10 μl of Splash Lipidomix mass spec standard were added. Cells were broken by glass bead beating and cell lysates transferred into Teflon lid screw glass tubes with the addition of 0.5 ml of CHCl3:CH3OH (2:1). Extraction proceeded for 30 min at room temperature with vigorous agitation. For lipid partitioning, 0.5 ml of CHCl3:CH3OH (5:1) and 1.5 ml of H2O were added, tubes subjected to vigorous agitation for 30 min at room temperature, and centrifuged at 3000 rpm for 10 min in an Allegra X-12R centrifuge using a swinging bucket SX4750 rotor. Around 0.5 ml of chloroformic bottom layer were removed and dried on a Speed Vac. Dried material was dissolved in 50 μl of lipidomic resuspension buffer  . Chromatographic separations of the lipid extracts were performed by injecting 10 μl of the lipid extracts on an Accucore C30 column  kept at 30 °C. The mobile phase composition and chromatographic conditions are shown in https://www.thermofisher.com/ca/en/home/industrial/mass-spectrometry/liquid-chromatography-mass-spectrometry-lc-ms/lc-ms-software/multi-omics-data-analysis/lipid-search-software.html) for lipid identification and quantitation. The data analysis parameters used are detailed in Yeast cells growing logarithmically were harvested, thoroughly washed with ice-cold HTaq DNA polymerase, Platinum Taq DNA polymerase High Fidelity, and pCR2.1-TOPO vector were from Invitrogen; Sea Kem Gold Agarose and Surfact-Amps X-100 were from Thermo Fisher Scientific; Benzonase was from Sigma; protease inhibitors cocktail Complete was from Roche. QuickChange mutagenesis kit was from Stratagene; amylose-resin, E. coli Express cells and restriction enzymes were from New England BioLabs; glutathione-Sepharose 4 Fast Flow was from Cytiva. Yeast nitrogen base without amino acids and without inositol was from Formedium. Synthetic complete drop-out mixes and yeast nitrogen base without amino acids and without ammonium sulfate were from Sunrise Science. Lipids and Splash Lipidomix mass spec standard were from Avanti Polar Lipids. Instant Blue Coomassie staining was from Abcam.All the data are contained within the manuscript or the This article contains The authors declare that they have no conflicts of interest with the contents of this article.


Although the noncanonical NFκB pathway was originally identified as a cellular pathway contributing to lymphoid organogenesis, in the past 20 years, its involvement in innate immunity has become more appreciated. In particular, the noncanonical NFκB pathway has been found to be activated and even exploited by some RNA viruses during infection. Intriguingly, activation of this pathway has been shown to have a role in disrupting transcription of type 1 interferon (IFN), suggesting a rationale for why this response could be co-opted by some viruses. Rift Valley fever virus (RVFV) is a trisegmented ambisense RNA virus that poses a considerable threat to domestic livestock and human health. Previously, we showed the atypical kinase RIOK3 is important for mounting an IFN response to RVFV infection of human epithelial cells, and shortly following infection with RVFV (MP12 strain), RIOK3 mRNA is alternatively spliced to its X2 isoform that encodes a truncated RIOK3 protein. Alternative splicing of RIOK3 mRNA has an inhibitory effect on the IFN response but also stimulates an NFκB-mediated inflammatory response. Here, we demonstrate alternative splicing of RIOK3 mRNA is associated with activation of the noncanonical NFκB pathway and suggest this pathway is co-opted by RVFV (MP12) to enhance viral success during infection. Careful co-ordination of interferon (IFN) and inflammatory responses to viral infection is essential for robust cellular immunity as well as avoidance of collateral tissue damage caused by overblown immune responses. Viruses have evolved many strategies to dysregulate discrete pathways to evade cellular responses to infection. Such dysregulation can be harmful to the host. For example, with the recent COVID-19 pandemic, exacerbated inflammatory responses coupled with a muted IFN response were frequently observed in the most severe clinical cases . TherefoBunyavirales, family Phenuiviridae, genus Phlebovirus [Aedes and Culex genera to humans and livestock, such as sheep, goats, and cattle [Rift Valley fever virus  is an ad cattle ,5,6. Humd cattle . Symptomd cattle ,9. Hepatd cattle ,11, and d cattle ,14,15,16d cattle ,18. UndeRVFV has an ambisense tripartite RNA genome and, following infection of a cell, genomic viral RNA is detected predominantly by the retinoic acid inducible gene I (RIG-I) intracellular receptor ,20. AfteAlthough the noncanonical NFκB pathway  was originally identified as a cellular pathway involved in adaptive immunity and secondary lymphoid organogenesis ,30,31,32Canonical  and noncanonical p52:RelB) NFκB dimers belong to the same family of NFκB transcription factors and share a Rel homology domain ,50,51,52:RelB NFκThere are several other characteristic differences between the canonical and noncanonical NFκB pathways. Typically, activation of the canonical pathway occurs almost immediately upon immune stimulation, but the noncanonical pathway has a much slower onset and depends on de novo protein synthesis ,33,62,63In addition to a type 1 IFN response that is initiated by RIG-I detection of viral RNA, both canonical and noncanonical NFκB pathways can also be activated when RIG-I associates with MAVS ,67. We a2; see text for specific times when cells were harvested post-infection. Quantification of infectious dose was performed in TCID50 assays described by Smith et al. [Vero 76, human embryonic kidney 293 (HEK 293), and hepatocarcinoma (HepG2) cells were obtained from ATCC . Vero 76 and HEK 293 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) and penicillin + streptomycin (pen/strep) , and HepG2 cells were grown in 1:1 DMEM:F12 containing 10% FBS and pen/strep (Thermo Fisher Scientific). Since renal failure and hepatic complications have frequently been observed in severe cases of RVFV infection, HEK 293 and HepG2 cells were selected for use in this study. Experiments were initiated when cells were near confluency (70–90%). Furthermore, the attenuated biosafety level 2 (BSL2) laboratory RVFV strain MP12  was used for all viral infections. MP12 is an attenuated RVFV strain derived from the wt ZH548 isolate, and this strain was used because it can be handled in BSL2 containment. RVFV MP12 harbors mutations in all three of its genomic segments to mitigate its pathogenicity , but manh et al.  with Ver2; see text for incubation times used in each assay.TNFα and LIGHT were purchased from BioLegend . Prior to treating cells with TNFα or LIGHT, growth media were removed; cells were washed with PBS, and PBS was exchanged with fresh DMEM media containing 10 or 2% FBS and pen/strep. Then, when treating with TNFα, TNFα was added such that its final concentration in each well was 20 ng/mL, and when treating with LIGHT, LIGHT was added to each well to reach a final concentration of 50 ng/mL. After treating with either reagent, cells were then incubated at 37 °C, 5% CO2. While the cells were incubating, transfection master mixes were prepared. When preparing Lipofectamine master mixes, Lipofectamine was added to OptiMEM media and incubated at room temperature for 5–20 min. Lipofectamine was added to each master mix such that the ratio of μL Lipofectamine 2000 to μg of construct transfected would be ~2.5 per well. After incubation of Lipofectamine master mixes, these mixtures were added to DNA construct master mixes comprising construct DNA and OptiMEM, and the resulting transfection mixture was left to incubate at room temperature for 20 min to allow assembly of DNA: liposome complexes. Transfection mixtures were then dispensed into wells with cells incubated in OptiMEM media, and the mixtures were added to each well at a volume equal to half the volume of growth media. Before any downstream treatments were initiated, cells were incubated with the transfection mixtures at 37 °C, 5% CO2 for at least 24 h to allow sufficient expression of the constructs. The RIOK3 X2 construct, which we previously showed expressed the predicted protein/peptide, was expressed in a phRL-CMV backbone, and the empty vector (EV) control plasmid described in the text was phRL-CMV backbone lacking any insert [Plasmid transfections were accomplished with Lipofectamine 2000 (Thermo Fisher Scientific). In these transfections, growth media were removed, cells were washed with PBS, and PBS in each well was exchanged with a volume of OptiMEM transfection media equal to half the volume of growth media. The cells were then incubated at 37 °C, 5% COy insert .g and 4 °C for 15 min. Aqueous layers containing RNA were then transferred to new tubes, and to precipitate the RNA, 2.5 μL of 4 mg/mL glycogen and 0.5 mL isopropanol per 1 mL of TRIzol used were added. After vortex and incubation at room temperature for 10 min, RNA samples were then spun at 12,000× g and 4 °C for 10 min. Supernatant was then removed, and the RNA pellets were washed with 1 mL of 75% ethanol per 1 mL of TRIzol used. Washes were accomplished by brief vortex of the pellets in 75% ethanol and centrifugation at 7600× g and 4 °C for 5 min. The 75% ethanol solution was then removed, and the pellets dried at room temperature for 5–10 min. RNA was then resuspended in nanopure H2O, incubated at 55–60 °C for 10–15 min, and stored at −80 °C or used immediately. Extracted RNA was then reverse transcribed to cDNA with Maxima H Minus Reverse Transcriptase (Thermo Fisher Scientific). Prior to reverse transcription, RNA in each sample was diluted to ~100 ng/μL. Diluted RNA was incubated with random hexamer primers and dNTPs at 65 °C for 5 min, chilled, and reverse transcription reagents (reverse transcription buffer and Maxima H Minus Reverse transcriptase) were then added. After addition of reverse transcription reagents, samples were incubated at 25 °C for 10 min., 50 °C for 30 min, and 85 °C for 5 min. Resulting cDNA was then either immediately used or stored at −20 °C.When extracting RNA, cells were lysed with TRIzol (Thermo Fisher Scientific), and RNA was extracted from the reagent according to the manufacturer’s protocol. To separate RNA into an aqueous layer, lysates were first treated with 0.2 mL chloroform per 1 mL TRIzol. Following vortex and incubation at room temperature for 10 min, the lysates were spun at 12,000× 2O. Measurements were obtained using a 384-well plate, and each well with sample contained 5 μL SYBR Green Master Mix, 1 μL forward + reverse 10 μM primer mix, and 4 μL diluted cDNA. cDNA was then amplified and measured under the following conditions: (1) 50 °C for 2 min, (2) 95 °C for 2 min, (3) 95 °C for 15 s, (4) 60 °C for 1 min, (5) plate read, (6) go to (3) 39×, (7) 65 °C for 31 s, (8) 65 °C for 5 s + 0.5 °C/cycle Ramp 0.5 °C/s, (9) plate read, (10) go to 8) 60×. Relative normalized expression was quantified in CFX Maestro (Bio-Rad) using the ∆∆Ct method, and RNA was normalized to GAPDH (see RT-qPCR measurements were obtained using SYBR Green Master Mix (Thermo Fisher Scientific) and the CFX Connect Real-Time PCR Detection System . First, cDNA was diluted 1:5 in nanopure H2O, and 2 μL cDNA. cDNA was then amplified under the following conditions: (1) 98 °C for 10 s, (2) 98 °C for 1 s, (3) 65 °C for 5 s, (4) 72 °C for 15 s, (5) go to (2) 29×, (6) 72 °C for 1 min, (7) 4 °C forever. Amplified cDNAs were then resolved on a 1% agarose gel at 80 V. After rocking gels for 10 min in a 1× TAE bath containing ethidium bromide, images were obtained using a Gel Doc XR + Molecular Imager (Bio-Rad). Figures show representative samples of biological duplicates or triplicates.Prior to electrophoresis, cDNA was amplified by PCR using InPhusion Flash Hi-Fidelity PCR Master Mix (Thermo Fisher Scientific). Each reaction mixture consisted of 10 μL Phusion Flash PCR Master Mix, 1 μL of 10μM forward primer, 1 μL of 10 μM reverse primer, 6 μL nanopure H2 for 2–5 min until they no longer adhered to the plate(s), and the suspensions were then transferred to conical centrifuge tubes and were centrifuged at ~1000 rpm, 4 °C for ~5 min. Trypsin was removed, and cell pellets were resuspended in chilled PBS (10 mL/cell pellet). Resuspensions were centrifuged at ~1000 rpm, 4 °C for 5–10 min, and PBS was removed. Cells were either lysed immediately or stored at −80 °C until lysis. Cells were lysed with radioimmunoprecipitation buffer , 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 140 mM NaCl) containing Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific). Cells harvested for Western blotting were grown in 6-well plates. Prior to harvest, media was removed, and the cells were washed with PBS (Thermo Fisher Scientific). To remove cells from the plate, cell scraping or trypsin (Thermo Fisher Scientific) was used. When scraping cells, after PBS wash, 5 mL PBS were added per well while the 6-well plate was on ice. Cells were scraped, and suspended cells were transferred to conical centrifuge tubes kept on ice. A total of 5 mL of PBS were again added per well, and scraping and transfer of suspensions to centrifuge tubes was repeated. The cells were then centrifuged at ~1000 rpm, 4 °C for 5–10 min. PBS was then removed, and the cells were either lysed immediately or stored at −80 °C until lysis. When removing cells from 6-well plates with trypsin, 1 mL trypsin was added per well after washing with PBS. Cells were incubated at 37 °C, 5% COw/v non-fat dry milk in TBST) at room temperature for 1 h and subsequently washed with Tris-buffered saline Tween-20  3 times, 5 min each time. Incubation in 1° antibodies was then performed at room temperature for 2 h or 4 °C overnight. TBST washes were again performed 3 times, 5 min each time, and the membrane was incubated with horseradish peroxidase (HRP) 2° antibodies at room temperature for 1 h. After washing with TBST 3 times, 10 min each time, membranes were incubated in visualization solution  and visualized on an Image Reader LAS-3000 . When visualizing proteins specific to more than one 1° antibody, the membrane was severed at a molecular weight distinguishing the two proteins and incubated separately with distinct 1° antibodies. The 1° antibodies used were rat anti-p100/p52, mouse anti-IκBα, mouse anti-β-tubulin, and mouse anti-GAPDH and were all purchased from BioLegend. The 2° antibodies used were HRP goat anti-rat IgG (BioLegend) and HRP goat anti-mouse IgG . Where applicable, band intensities were quantified by ImageJ software [Lysates were either used immediately or stored at −80 °C. Total protein concentrations in lysates were then measured with a BCA assay (Thermo Fisher Scientific) and diluted accordingly with RIPA to reach as equal concentrations as possible. Proteins were then resolved by denaturing polyacrylamide gel electrophoresis using Tris-Glycine SDS Running Buffer  and 8% acrylamide Tris-Glycine precast gels . Proteins were transferred from the gel to PVDF membrane  at 30 V for 960 min in transfer buffer . Membranes were blocked with non-fat milk  control plasmid, the increase was not statistically significant . HoweverFurthermore, to assess whether RIOK3 X2′s engagement of the noncanonical NFκB pathway during RVFV MP12 infection affects the IFN response, relative expression of IFNβ was also measured in MP12-infected HEK 293 cells overexpressing RIOK3 X2. Compared to infected cells transfected with control plasmid, there was a statistically significant decrease in IFNβ expression in infected cells overexpressing RIOK3 X2 b. Again,Lastly, to directly determine whether the effects of RIOK3 X2 on innate immune responses could be beneficial for RVFV propagation in cell culture, virus titers in supernatants of MP12-infected HEK 293 cells transfected with RIOK3 X2 or MP12-infected HEK 293 cells transfected with control plasmids were quantified by TCID50 assays. TCID50/mL was ~2 logs higher in supernatant from infected cells overexpressing RIOK3 X2 compared to that of controls c. TherefA hallmark of the noncanonical NFκB pathway is the appearance of p52, the proteolytically processed subunit of p100. Cellular expression of p52 is exquisitely under tight regulation by the cell, and even small changes in its steady-state levels are associated with activation or suppression of the noncanonical pathway ,78. Whenbefore activation of the canonical NFκB pathway during RFVF MP12 infection, which would not be expected if the activation of the noncanonical pathway we observe was simply part of a coordinated innate immune response to the virus. That is, in a typical cellular innate immune response to viral infection, activation of noncanonical NFκB pathway would be expected to occur significantly later than activation of the canonical pathway. Furthermore, elevated infectious particles observed in RIOK3 X2 overexpressing cells that were shown to have elevated p100 expression further suggest the supportive role this pathway could have in RVFV replication and spread.Since the noncanonical NFκB pathway has an inhibitory effect on the antiviral IFN response, exploiting and co-opting this pathway was proposed to be advantageous for some viruses ,36,37. OHere, we add RVFV to the short list of viruses that use this unusual strategy to enhance their success. Furthermore, we demonstrate that alternative splicing of key genes is instrumental in co-opting the noncanonical NFκB pathway during RVFV infection. Exactly how alternative splicing of RIOK3 is involved in the cell’s activation of the noncanonical NFκB pathway and how RIOK3 X2 expression results in increased expression of p100 will need to be addressed in future studies. However, one potential mechanism for how RIOK3 X2 might be able to increase p100 expression could be through RIOK3 X2 mediating crosstalk between the canonical and noncanonical pathways. Full-length RIOK3 has been shown to inhibit the canonical NFκB pathway ,86, whilIt is still unclear whether alternative splicing of RIOK3 alone can activate the noncanonical NFκB pathway or whether RIOK3 alternative splicing occurs downstream of the initial activation of this pathway. Here, we observed alternative splicing of RIOK3 after p52 begins to appear at 24 h post-TNFα treatment, so it is possible that alternative splicing of RIOK3 occurs after the initial activation of the noncanonical pathway. Moreover, alternative splicing in general has been shown to be important for regulating both canonical and noncanonical NFκB pathways ,88, and The present findings help illuminate how cellular innate immune pathways can be targeted and co-opted for viral success . We show


Alkalihalobacillus clausii, Bacillus safensis, and Escherichia coli.Here, we present the coding-complete genomes of 11 lytic bacteriophages isolated from bovine ruminal fluid and vaginal swabs that can infect the bacterial hosts  Cattle origin, sample collection, and bacterial host isolation were described previously  were suspended in phosphate buffered saline and centrifuged at 12,000 × g for 15 min at 4°C. Supernatants were passed through a 0.22-µm filter and used for a spot assay. Overnight bacterial host cultures (100 µL) were inoculated into 3 mL of brain heart infusion (BHI) soft agar (0.5% agar) which was then poured on top of BHI agar plates. Then, 10 µL of the filtered supernatant was spotted on top agar and incubated overnight at 37°C with 5% CO2. Areas of lysis were transferred into 1 mL of SM buffer (G-Biosciences), centrifuged at 20,000 × g for 1 min. The supernatant was then filtered using a 0.22-µm filter and stored at 4°C. Next, 100 µL of overnight host bacterial culture and 100 µL of phage stock were added to 3 mL of soft BHI agar, poured onto BHI plates, and incubated overnight at 37°C with 5% CO2. Phages were purified by two successive rounds of plating with the bacterial hosts.Eleven bacteriophageeviously , 2. For https://github.com/tseemann/shovill). Qualities were assessed with QUAST  according to manufacturer’s instructions with an additional initial step in which DNase and proteinase K were used to remove host genetic material . Genomicth QUAST , assemblth QUAST , and scrth QUAST , VFDB /genome length. None of the genomes contained CRISPRs, tmRNAs, virulence factors, or antimicrobial resistance genes. Genomes were compared to those in the GenBank database using BLASTn. Sequence similarity (% identity × % coverage) was used to determine taxonomy. The identities of ≥95%, 70%–95%, or <70% were considered as the same species, genus, or family, respectively  Table 1Table 1.


This paper presents the resultsof structural, optical, magnetic, and electrical properties, as wellas the broadband dielectric spectroscopy measurements of single crystalswith different Nd3+ ion concentrations, i.e., when x = 0.0050 or x = 0.0098,and with constant content of Mn2+ ions, i.e., when y = 0.0050. Our magnetic studies have shownthat substitution of diamagnetic Ca2+ ions in the CaMoO4 matrix with paramagnetic Nd3+ ones with a contentnot exceeding 0.02 and having a screened 4f-shell revealed a significanteffect of orbital diamagnetism and Van Vleck’s paramagnetism.Both single crystals have revealed residual electrical conductivitywithout an intrinsic region and a change of sign of the Seebeck coefficientat ca. 230 K. Dielectric spectroscopy measurements have shown constantvalues of relative permittivity (εr ∼ 8) andloss tangent (tan δ ∼ 0.01) both up to 400 K and up to1 MHz, as well as the Fermi energy (∼0.04 eV) and the Fermitemperature (∼500 K) determined for both crystals from thediffusion component of thermopower. These results suggest the presenceof shallow acceptor and donor levels in the studied crystals.Single crystals of Ca I41/a), both undoped as well as activated with d- or f-electron metalions, are important materials because of their interesting luminescentand magnetic properties. They are already used as, e.g., efficient phosphors,2 solid state lasers3 scintillators,5 microwave dielectrics,7 and catalysts.8 The combination of severaldoping ions  is an increasingly popular and promisingway of obtaining new materials with even more interesting propertiesand wide industrial applications, e.g., ceramicssuch as CaSO4/Dy3+, Mn2+, or MgB4O7/Tb3+, Mn2+. These micropowders,when irradiated with γ-radiation, show 2–4 times moreintensive thermoluminescence than the materials currently used inionizing radiation dosimeters.10Divalent metal molybdates and tungstateswith the tetragonal scheelite-typestructure (space group 4) and calcium tungstate, as members of the scheelite family,are highly ionic compounds with a small contribution of covalent bonding.Band gaps determined from the band dispersion calculations indicatedthat both compounds are direct band gap insulators, with values of3.41 and 4.09 eV for CaMoO4 and CaWO4, respectively.11Calcium molybdate, characteristic of shallowdonor levels.24 Summarizing our resultson a wide group of scheelite-type materials (micro- or nanopowders)codoped with divalent d-electron metal ions and trivalent rare-earthmetal ones, it can be stated that paramagnetism is dominant everywhere,mainly due to the screening of electrons in the 4f subshells of RE3+ ions. For the samples poorer in magnetic ions, magneticcontributions independent of temperature, such as orbital diamagnetismor Van Vleck paramagnetism in nonmetals, played an important role.Generally, doped scheelite-type single crystals for such matricesas CdMoO4, CdMoO4/WO4, and PbMoO4/WO4 show semiconducting properties. Dipole relaxationprocesses and colossal dielectric permittivity were only observedfor a CdMoO4/Eu3+ single crystal.Our previous studies of calcium molybdatenanomaterials doped withMnx–y1–3Mny□xNdx2(MoO4)x1–3(WO4)x3  with very similar Mn2+ and Nd3+ concentrations as the single crystals under study was synthesizedby the solid-state reaction method and studied.25 These studies revealed a scheelite-type structure of obtainedmicropowders, a direct band gap in the center of the Brillouin zonechanging nonlinearly with an increase in the content of Nd3+ ions in the range of 3.70–3.83 eV, a significant effect oforbital diamagnetism and Van Vleck’s paramagnetism for samplespoorer in neodymium ions, a paramagnetic behavior with long-rangeferrimagnetic (FIM) and short-range antiferromagnetic (AFM) interactions,weak n-type electrical conductivity characteristic for insulators,an extrinsic region with low activation of 0.004 eV below 300 K andan intrinsic one with stronger activation of 1.0 eV above 300 K, andsmall values of both relative permittivity (∼12) and loss tangent(∼0.01), which were weakly dependent on temperature and frequency.25Recently,the solid solution of Cax–y1–3Mny□xNdx2(MoO4)x1–3(WO4)x3 single crystals , obtained by theCzochralski method, were systematicallyinvestigated and compared with the microcrystalline materials of thesame chemical formula. The study of poorly conductive and highly magneticallydiluted single crystals and the impact of technology on their propertiesis a novelty of this work.In this study, Cax–y1–3Mny□xNdx2(MoO4)x1–3(WO4)x3 solid solution  were successfully grown by the Czochralski method in aninductively heated iridium crucible in nitrogen as a protective atmosphere.The starting materials for the crystallization processes of both singlecrystals were: CaCO3 , MnO, Nd2O3 ,MoO3 , and WO3 . The stoichiometric amounts of initial reactants wereground homogeneously in an agate mortar, pressed into cylindricalpellets under pressure of 200 kPa, and heated at 800 °C for 12h in the air before melting in an iridium crucible. Then, the microcrystallinematerials were heated to a temperature of ∼50 °C higherthan their melting point. Thus, the obtained melts were maintainedunder these conditions for 2 h. Next, the temperature was decreasedto the appropriate crystallization point. The single crystals of calciummolybdato-tungstates codoped with Nd3+ and Mn2+ ions were grown on the [001]-oriented seeds prepared from a singlecrystal of CaMoO4. The as-grown crystals were dark bluein color  single crystal—Ca 19.35(4) mas % ; Mn 0.13(1) mas % ; Nd 0.67(2) mas %; Mo 46.64(6) mas % ; W 1.39(2)mas % ; the CMNMWO  single crystal—Ca 18.87(4) mas % ; Mn 0.12(1) mas % ; Nd 1.33(4) mas %; Mo 45.49(7) mas % ; and W 2.60(3)mas % . The determined contents of Ca, Mn, Nd, Mo,and W in both CMNMWO single crystals closely corresponded to the proposedchemical formulas, i.e., when x =0.0050 or x = 0.0098 and y = 0.0050.The density of both CMNMWO crystals was determined using a QuantachromeInstruments Ultrapycnometer . Nitrogen(purity 99.99%) was applied as a pycnometric gas. The values of densitywere found to be 4.34(2) g cm–3 (x = 0.0050) and 4.35(2) g cm–3 (x = 0.0098). Small pieces of both CMNMWO single crystals obtainedafter cutting out the plates were ground in an agate mortar. The obtainedmicrocrystalline powders were examined by a powder XRD method usingan EMPYREAN II diffractometer  and Cu Kα1,2 radiation (λ = 1.5418 Å). XRD patterns were collectedin the 10–100° 2Θ range with a scanning step of0.013° and analyzed by HighScore Plus 4.0 software. Lattice constantswere calculated using DICVOL04 software.26I41/a (JCPDs No.04-013-6763).Single crystals of Cain color 1a.They in color 1b. Such-Pro27 programwas used for the collection of the data as well as for determinationand refinement of the unit cell parameters from about 4000 reflections.Integrations of the collected data were also performed using CrysAlisPro.27 SHELXL-97 program28 was used to refine the structure of both singlecrystals. The positions of O atoms and the anisotropic displacementparameters of all atoms were refined.A SuperNova kappa diffractometer, equippedwith a Mo Kα X-ray tube and Atlas CCD detector (Agilent Technologies),was used for the XRD measurements, which were performed at 293(1)K. The CrysAlisUltraviolet and visible(UV–vis) diffuse reflectance spectroscopy was realized witha JASCO-V670  spectrophotometerequipped with an integrating sphere. The spectra were recorded inthe range of 200 to 1000 nm with a scanning rate of 0.5 nm.eff moment was determined using the 30k is the Boltzmannconstant, NA is the Avogadro number, μB is the Bohr magneton, and C is the molarCurie constant. The effective number of Bohr magnetons peff was calculated from the eq 3+  and Mn2+  ions with 4f3 and3d5 electronic configuration, respectively.The static (DC) magnetic susceptibility was measured in the temperaturerange of 2–300 K and recorded in both zero-field-cooled (ZFC)and field-cooled (FC) modes. Magnetization isotherms were measuredat 2, 10, 20, 40, 60, and 300 K by using a Quantum Design MPMS-XL-7ACSQUID magnetometer  in appliedexternal fields up to 70 kOe. The effective magnetic μq 2where  for Nd3+T), was measuredby the DC method using a KEITHLEY 6517B electrometer/high-resistancemeter . The thermoelectricpower S(T), i.e., the Seebeck coefficient, was measured in the temperature rangeof 100–400 K with the help of a Seebeck Effect MeasurementSystem .Electricalconductivity, σwithin the temperature range of 76–400 K and in the frequencyrange from 5 × 10I41/a space group, similarly to some divalent molybdates andtungstates, i.e., AMo(W)O4 .10 The unit cell parametersof the CMNMWO crystal  are as follows: a = b = 5.22766(10) Å and c = 11.4330(3) Å.The R value is equal to 0.0149. In the case of theCMNMWO single crystal , the lattice constants are as follows: a = b = 5.23207(12) and c = 11.4424(5)Å. The R value is equal to 0.0164. The mostimportant crystallographic data for both single crystals are collectedin Tables S1–S8. The calculatedlattice parameters of both crystals increased with the increase inNd3+ ions concentration. This is not a typical behaviorbecause bigger Ca2+ ions in the CaMoO4 matrixwere simultaneously substituted by much smaller Nd3+ andMn2+ ones (r (Ca2+) = 1.12Å > r (Nd3+) = 1.053 Å > r (Mn2+) = 0.96 Å for CN = 8).32 In scheelite-type molybdates and tungstateswhen r (A2+) > 0.9 Å, molybdenumand tungsten ions are tetrahedrally coordinated by O2– ones, and their ionic radii are very similar, i.e., they are 0.41 and 0.42 Å, respectively.32 For this reason, no significant changes in the latticeparameters of both CMNMWO single crystals should be observed whenMo6+ ions are substituted by W6+ ones. The crystallattice expansion observed for CMNMWO single crystals is also visibleafter grinding these samples. With the increase of Nd3+ content, all diffraction lines observed in the XRD patterns of powdermaterials shifted toward the lower angle direction (x = 0.0050) a = b = 5.22731(10) and c = 11.4296(4) Å;CMNMWO (x = 0.0098) a = b = 5.23096(11) and c = 11.4396(6) Å.The XRD studies revealed that bothsingle crystals belong to tetragonal symmetry and crystallize withscheelite-type structure in the irection 1c. In or33The optical properties of CMNMWOsingle crystals along both crystallographic directions were investigatedat room temperature by using the UV–vis diffuse reflectancespectroscopy method. This technique enables the determination of suchimportant optical parameters of materials as the band gap energy,absorption coefficient, and others. The theory which makes possiblethe use of diffuse reflectance spectra for solids was proposed byKubelka and Munk.33F(R) is the Kubelka–Munk approach, R is thereflectance, α is the absorption coefficient, and S is the scattering factor, which is wavelength-independent. The opticalband energy gap (Eg) is related to theabsorbance and photon energy in the 35hν is the photonenergy, A is an energy-independent constant characteristicof a material, and n is a constant that can takedifferent values depending on the nature of the electronic transition.The permitted direct, forbidden direct, permitted indirect, and forbiddenindirect transitions take place when n = 1/2, 3/2,2, and 3, respectively.35 According to the literature,scheelite-type materials such as CaMoO4 and CaWO4 show an optical spectrum regulated by the direct absorption process, i.e., when n = 1/2.10 In the low-energy region, the absorption edge (αhν)2 changed linearly with the energy ofthe photon. Thus, in the high-energy region, the absorption spectrumdeviated from the straight-line plot. This straight-line behaviorin the low-energy region was considered the main evidence of a directoptical bandgap. x = 0.0098 in the wavelength from 200to 1000 nm. The intense and very broad band is observed in the spectralrange of 300–700 nm for this sample. A similar broad band isalso observed for the CMNMWO single crystal with a lower concentrationof Nd3+ ions (not shown here). In this spectral range,the following charge transfer bands occur: O2– →Mo6+, O2– → Mo5+ presentin small amounts in as-grown CMNMWO single crystals, O2– → W6+, O2– → Nd3+ as well as O2– → Mn2+.38 In the visible range, the bands associated with 4f–4f transitionsof Nd3+ are also observed.40x = 0.0098)for the [001] crystallographic direction deconvoluted into Gaussiancomponents with the assignment of bands for Nd3+ ions inthe visible region.According tothis method, the reflectance spectra can be converted into absorptionones using the hν)2 curve obtained for both crystals as well asfor [100] and [001] crystallographic directions to the photon energyaxis  as well as 3.71 eV , as they affect the magnetic parameters of the single crystalsunder study. In general, these contributions significantly changethe value of the magnetic susceptibility in the Curie–Weissregion by the factor χ0, which represents all temperature-independentsusceptibilities.41 The χ0 factor was determined from the linear function χT(T) in the Curie–Weiss region from the 24b is the intercept,and χ0 is the slope. The dependencies of productχZFC·T(T)from the measurement and the asymptotes determined from b and χ0 are shownin The results of magnetic measurementsof the CMNMWO single crystals are depicted in 0 = 0) for a single crystal with x = 0.0050, while orbital diamagnetism (χ0 < 0)dominates for a single crystal with x = 0.0098. Thismeans that for a larger amount of neodymium ions in the sample, VanVleck paramagnetism weakens. Both single crystals shown in ZFC(T)-curve downwardfrom its linear part as well as the short-range AFM interactions visiblein negative values of the Curie–Weiss temperature x1–3(WO4)x3 solid solution (x = 0.0010 and 0.0050),24 it was observedthat the values of the effective magnetic moment (μeff) are much higher than the effective number of Bohr magnetons (peff).No splittingbetween ZFC and FC magnetic susceptibilities suggestsno spin frustration. The effective magnetic moment, μThis difference was explained bythe fact that some molybdenumions may be in an oxidation state lower than +6, contributing to anincrease in the net magnetic moment.25 of the same solid solution asthe single crystals under study, as well as for single crystals ofPbx1–3□xNdx2(MoO4)x1–3(WO4)x3 solidsolution.24 The reason for this could bea magneto-crystalline anisotropy.The results of the magneticmoment measurements of CMNMWO singlecrystals are shown in 3/T), of the CMNMWOsingle crystals showed residual electrical conductivity without intrinsicregion in the temperature range of 77–400 K and the value ofelectrical conductivity at 400 K of only 2.7 × 10–9 S/m , was also observed among others in Cax1–Mnx(MoO4)0.50(WO4)0.50 ceramics,13 Cax1–MnxMoO4 nanomaterials,12 CdRE2W2O10 tungstatemicrocrystals ,42 and Cax–y1–3Coy□xGdx2(MoO4)x1–3(WO4)x3 molybdato-tungstate micropowders.14 Residual electrical conductivity of the p-type up to 230 K and then-type above this temperature (n) are always present in the lattice even in a crystal whichis ideal in other respects. A necessary condition for free energyminimalization gives n ≃ N exp (−EV/kT)for n ≪ N, where N is the number of atoms in the crystal, and EV is the energy required to transfer the atom from thebulk of the crystal on its surface.43No intrinsic region in the relation, σ(10perature 7 in the S(T), shown in i.e., the diffusion contribution (Sdiff), which is proportional to temperature according to theMott formula,44 and the phonon drag contribution(Sph), which is more complex and resultsfrom the transfer of phonon momentum to the electron gas. The Sph component has an extreme in the range , where θD isthe Debey temperature.45 The θD value estimated from the Debye formula24 is 349 K for both single crystals. The S(T) maximum observed below 230 K in 44e is the elementarycharge, EF is the Fermi energy, and a is an empirical slope. From EF, can be written as followsThe temperature dependence of the thermoelectricpower EF and Fermi temperature TF (definedas EF/k) are summarizedin e.g., for pure copper: EF = 7 eV and TF = 8.19 ×104 K43 and to non-metallicconductors, e.g., for Cux1–GaxCr2Se4 single crystals: EF ∼ 0.3 eVand TF ∼3 × 103 K,46 as well asto Pbx1–3□xNdx2(MoO4)x1–3(WO4)x3 singlecrystals: 0.036 ≤ EF ≤ 0.048eV and 418 ≤ TF ≤ 557 K,24 it can be seen that the latter have very smallvalues, such as the single crystals under study. The Fermi level inthe studied single crystals with EF =0.04 eV compared to Eg = 1.7 eV meansthat the EF is close to the border ofthe valence band, similar to the case of insulators. This means thatthe studied single crystals are in fact intrinsic semiconductors,in which the conductivity is half and half electron–hole. Therefore,an increase in electrical conductivity up to 400 K is not observedbecause electrons cannot increase their energy without jumping intothe conduction band.The values of r, and the loss tangent,tan δ, for the various electric field frequencies of the CMNMWOsingle crystals. As one can see, for each single crystal, the valueof the permittivity remains almost independent of the frequency, andit only slightly increases with temperature. However, this increaseis very minor, and, e.g., for the single crystalwhen x = 0.0050, εr changes from9.1 at 80 K up to 9.7 at 401 K . This suggests that in the single crystals understudy, no dipole relaxation processes were observed in the temperaturerange up to 400 K. Therefore, the accumulation of charge visible inthe spectra may be caused by space charge polarization, as the studiedsingle crystals are practically intrinsic semiconductors (insulators).Temperature dependences of εr(T)and tan δ(T) do not also indicate an occurrenceof dipole relaxation processes or energy losses at low frequenciesinduced by electric conduction, i.e., so-called Joule-Lenzlosses. Materials with such low energy loss can be successfully usedin the fabrication of lossless capacitors. Similarly, a low energyloss was observed in vacancied lead tungstates with high Pr3+ ion content.21at 401 K 8a. Also,at 401 K 8b. For t0098, εr 8c and ta0098, εr 8dhave s2+ site substitutionby Nd3+ and Mn2+ ions on structural, optical,magnetic, and dielectric properties in CMNMWO single crystals (where x = 0.0050 as well as x = 0.0098 and y = 0.0050) was studied. XRD analysis identified that boththe CMNMWO crystals crystallize in a tetragonal scheelite-type structure(space group I41/a).The direct optical band gap was found to be ∼1.70 eV for bothsingle crystals and both crystallographic directions. The CMNMWO singlecrystals showed paramagnetic behavior with FIM long-range and AFMshort-range interactions and a significant effect of orbital diamagnetismand Van Vleck’s paramagnetism. The electrical studies revealeda residual electrical conductivity without an intrinsic region anda change of sign of the Seebeck coefficient at ∼230 K, constantrelative permittivity (∼8) and loss tangent (∼0.01)both up to 400 K and up to 1 MHz, as well as the Fermi energy (∼0.04eV) and the Fermi temperature (∼500 K) determined from thediffusion component of the thermopower characteristic of the presenceof shallow acceptor and donor levels. Compared to microcrystallinesamples of the same chemical formula, a weakening of electron transportwas observed in the studied single crystals due to the lack of thermalactivation up to 400 K. The Czochralski method, however, had no significanteffect on their magnetic and dielectric properties. The results presentedin our paper make the CMNMWO single crystals attractive candidatesfor optical sensors as well as thermoelectric devices and losslesscapacitors.In this study, the effect of Ca


We have found that the mixed phase of V7O16 and V2O5 can be stabilized by post-annealing of amorphous VxOy between 300–400 °C, which gets fully converted into V2O5 after annealing at higher temperatures ∼450 °C. The local conversion from VxOy to V2O5 has also been demonstrated by applying different laser powers in Raman spectroscopy measurements. The optical transmission of these films increases as the content of V2O5 increases but the electrical conductivity and the optical bandgap decrease. These results are explained by the role of defects (oxygen vacancies) through the photoluminescence (PL) and time-resolved photoluminescence (TRPL) measurements. The IR sensitivity of the mixed phase is explained by the plasmonic absorption by the V7O16 degenerate semiconductor.We report an infrared (IR) sensitive mixed phase of V 7O16 and V2O5 thin films for IR sensing and smart windows.Mixed phase of V However, these materials need to be cooled at lower temperatures for these applications. Vanadium-based oxides, found in more than 52 stable and metastable phases, have been investigated for developing uncooled devices as they show metal–insulator transitions (MIT) close to room temperatures under different external stimuli such as thermal, electrical, and optical.8–14 Vanadium (V) can have oxidation states varying from +2 to +5, where V2O3, VO2, and V2O5 are the most studied phases but other non-stoichiometric phases such as V3O7, V4O9, V6O13, V7O16 have also been reported, that occur due to the presence of more than one valency of V atom in a single crystal structure.14–17 Although, VO2  has been the most preferred phase for IR sensors10,11,18 and smart windows due to sharp MIT close to room temperature,19–22 it has limitations of growing in the right phase on arbitrary substrates at low temperatures. On the other hand, V2O5 having a layered structure is important for energy storage electrodes,23–26 gas sensors27–29 and chromogenic applications.30–32 However, the applicability of the pure V2O5 phase is again largely restricted due to high temperature growth and poor electrical conductivity. The mixed phase of VO2 and V2O5 has been reported to give enhanced IR sensitivity.33 Other phases such as V7O16 could be promising for these applications which has the mixture of both V4+ and V5+ arranged in a layered crystal structure.15,34,35 However, there are very few reports on V7O16 phase which has been reported during chemical synthesis of V2O5 nanotubes28,35 or in thin film form when grown under oxygen deficient conditions using pulsed laser deposition29 and atomic layer deposition.15 There is a clear research gap for in-depth understanding of electronic and optical properties of V7O16 for any practical application in smart windows and IR sensors. Here, we systematically tune the contents of V7O16 and V2O5 in a mixed phase  by post-annealing of amorphous vanadium oxide thin films grown by cathodic vacuum arc-deposition. We report a strong modulation of electrical resistance and optical transmission in these films with IR radiation and temperature.Infrared (IR) sensitive materials have been of significant interest for IR sensors and smart windows in which the resistance or the optical transmission can be modulated by heating or IR radiation.−3 mbar, arc current of ∼150 A, substrate temperature ∼200 °C and substrate bias of −60 V. These films were then taken out and annealed in air at different temperatures i.e., ∼300 °C, ∼350 °C, ∼400 °C, ∼450 °C, and 520 °C for 2 hours. The unannealed sample will be called as Sas-grown and other annealed sampled as S300, S350, S400, S450, and S520 in this manuscript. The Raman spectroscopy was carried out using a commercial spectrometer by WiTech (Alpha 300) having a green laser of 532 nm wavelength. The crystal structure was confirmed by X-ray diffraction  by scanning 2θ ranging from 10° to 90° at a step size of 0.02° using Cu Kα radiation (λ ∼ 1.54 Å). The X-ray photoelectron spectroscopy (XPS) was carried out using XPS/ESCA, K-ALPHA+, Thermo Fisher Scientific. The deconvolution and fitting of XPS data of was performed using XPSpeak41 software. The optical transmission spectra were obtained in the wavelength range from 380 nm to 800 nm using a UV-visible spectrometer by PerkinElmer LAMBDA 365 model. Electrical measurements were performed using a four-probe setup equipped with Keithley electrometers. The IR sensing measurements were carried out using the IR source by Newport model 6363IR having intensity ∼438 W m−2. Photoluminescence (PL) and time-resolved photoluminescence (TRPL) were recorded at the excitation wavelength of ∼350 nm and ∼266 nm (pulsed) using Edinburgh (FLS 980D2D2) set-up.First an amorphous vanadium oxide thin films were grown on high-grade barium-borosilicate 7059 glass substrates using cathodic vacuum arc-deposition by evaporating a vanadium metal target (∼99.99% purity) in the presence of oxygen at a partial pressure of ∼1 × 10as-grown, S300, S350, S400, S450, and S520 are shown in as-grown to sharp peaks in annealed samples at ∼15.3°, ∼20.18°, ∼21.65°, ∼31.04°, ∼41.21° and ∼41.87° related to (020), (001), (011), (040), (002), and (012) crystalline planes of the pure orthorhombic phase of V2O5 respectively. However, the peak at ∼24.5° confirms the presence of the V7O16 triclinic phase15,34 in S350 and S400 samples.The XRD patterns of S7O16, the X-ray photoelectron spectroscopy (XPS) were performed on S300 and S520, as shown in 3/2 and 2p1/2 due to the spin–orbit splitting of ∼7.5 eV. However, each peak 2p3/2 and 2p1/2 were further deconvoluted that correspond to the presence of V4+ (∼516.5 eV & ∼524 eV) and V5+ (∼517.5 eV & ∼525 eV).15,36 The peak at ∼530 eV is related to 1s oxygen in vanadium oxygen bonds,37,38 but other peaks at higher energies ∼532 eV are due to free hydroxyl groups adsorbed on the hydrophilic surfaces.39 The ratio of V4+ and V5+ contents in S300 and S520, estimated by the dividing the area under the curve of respective peaks, is found to be consistent with literature,15,40 implying higher V4+ content in these samples having V7O16 phase. In S520, peaks corresponding to V4+ were observed because of the photoreduction of V5+ in V2O5 phase.41To further confirm the presence of Vas-grown film, no sharp peaks were observed but as annealing temperature exceeds 300 °C, sharp peaks start appearing. The Raman spectra for S450 and S520 shows peaks at ∼101, ∼144, ∼194, ∼282, ∼304, ∼404, ∼483, ∼507, ∼528, ∼699, and ∼992 cm−1 due to different vibrational modes of V2O5 phase,14,15,42–44 as description given in −1 arises from the V–O–V chains of the layered structure of α-V2O5, as shown in 300 and S350), the peaks around ∼158, ∼255, ∼294, ∼832, ∼870, ∼970 cm−1 confirms the presence of V7O16 phase, also consistent with the earlier studies.14,44,45 It is to be noted that the presence of other polymorphs of V2O5 (β- γ-)14,26,42,44 cannot be completely ruled out using Raman spectroscopy due to overlapping peak positions.147O16 phase where the layered structure resembles to α-V2O5, causing the peak ∼144 cm−1 to be shifted to higher wavenumber ∼158 cm−1. However, the other vibrational modes related to ladder steps (LS) seems to be missing in V7O16, indicating the absence of LS in this phase. Clearly, other peaks at ∼255, ∼294, ∼832, ∼870, and ∼970 cm−1 could be assigned to the vibrational modes of V PBM data was replaced with SVG by xgml2pxml:<glyph-data id="z.dbd" format="PBM" resolution="300" x-size="8" y-size="10" xml:space="preserve">00000000000000000000000000000000111111110000000011111111000000000000000000000000</glyph-data><svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="13.200000pt" height="16.000000pt" viewBox="0 0 13.200000 16.000000" preserveAspectRatio="xMidYMid meet"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform="translate scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>O bonds (Ag symmetry).15 Also, the Raman spectroscopy performed at different laser powers (5 mW to 10 mW) in 2O5, unlike the annealed samples. Similar conversion was also observed by other groups in case of chemically synthesized VOx nanotubes.43 This study opens further questions of stabilizing the intermediate phase of V7O16 using laser heating and open the opportunities for applications in laser writing for high security tags and detection 350 and S520 samples. Since the Raman spectra of the S350 show the characteristic peaks at ∼832 cm−1, 870 cm−1, 920 cm−1 and S520 at 994 cm−1, we performed Raman mapping by selecting two wavelengths range from 750–910 cm−1 and 950–1050 cm−1 so that we could capture the regions of V7O16 and V2O5 spatially in 10 × 10 μm2 area. In 350 both V7O16 and V2O5 phases can be seen. On the other hand, for S520 the brighter region mostly cover the V2O5 with some minor black regions due to some other phases.The Raman spectroscopic results of these films are compared in as-grown, S300, S350, S400, S450, and S520. Apparently, the transmittance gradually increases with increasing the annealing temperature, and the maximum transmittance was observed in sample S400. The optical band gap was calculated for all the samples by using Tauc's equations.46 The calculated energy band gap of these samples decreases from ∼2.85 eV to 2.2 eV for the samples annealed at higher temperatures [eratures .Eu) shown in as-grown sample clearly having more defects shows highest Eu value which decreases by increasing annealing temperature, that indicate the defects are significantly reduced after annealing. The electrical conductivity (measured by four-probe) also decreases for the samples annealed at higher temperatures, as shown in Y-axis, having clear correlation with the electronic properties. The results are explained by considering a band-model shown in 7O16 is higher than the V2O5, it still has high conductivity due to the presence of more V4+ content, that contribute extra electrons in 3d conduction band. Also, the amorphous and low-temperature annealed samples are expected to have large number of defects and oxygen vacancies, that can even push the Fermi level into the conduction band, making V7O16 a degenerate semiconductor.23 Therefore, the overall conductivity of mixed phase is higher than the V2O5 due to the presence of V7O16. On the other hand, the samples annealed at high-temperature are defects free, despite having a lower bandgap and the Fermi level lies in the band gap. The dynamic resistance versus temperature measurements also confirms the metallic behavior of mixed phase, where the resistance is found to increase with temperature compared to pure V2O5 showing a typical decrease in resistance with temperature of semiconducting behavior, as shown in ESI Fig. S1.Usually, the amorphous semiconductors exhibit band-tailing due to the structural disorder which can be quantified as Urbach energy  and time-resolved-photoluminescence (TRPL) results of Sas-grown, S350, and S450. In TRPL, the peak intensity decay (ITRPL) with time (t) can be fitted using the following equationwhere Ai, τi are constants representing the amplitude and the lifetime of carriers. Here, we have used component i = 3 to fit the experimental curve and calculated the average decay time using the following expressionTo further investigate the nature of defects, we have measured the carrier life using TRPL as shown in τ1 and τ2 attributes to the fast decay through trap-mediated recombination, while τ3 corresponds to slow decay through radiative recombination.37 The average decay time of S450 is higher compared than other two samples due to the reduction in oxygen vacancies in the samples, which agrees well with the previous observations.48 These results are in good agreement with the Urbach energy measurements shown in The extracted fitting parameters and average decay time are shown in Finally, we demonstrate the sensitivity of our mixed phase sample by heating and IR radiation as shown in 7O16 being a degenerate semiconductor could also be suitable candidate for NIR plasmonics. Indeed, the broad peak ∼650 nm in the UV-vis spectrum could be due to the NIR plasmons absorption.23 Since the plasmonic frequency  is related to the carrier concentration (n) and the effective mass (m*), where e and ε0 are electronic charge and the permittivity of free space, respectively. The measured value of resonant frequency ∼4.6 × 1014 Hz gives the carrier concentration ∼1020 to 1021 cm−3, which is also consistent with our conductivity values considering the theoretical values of mobilities and effective mass of vanadium oxides (1–1.7me).49,50 The IR sensitivity in optical transmission is due to the plasmonic absorption and consistent with the earlier studied on the mixed phase of metallic VO2 and insulating V2O5.33 Also, the increase in resistance under IR radiation seen in versus temperature measurements. Clearly, more low temperature transport measurements are required to further investigate the transport properties, but these results are encouraging for their promising use in smart-windows and IR sensors.Also, V7O16 and systematically tune the electronic and optical properties of a mixed phase by changing the V4+ and V5+ contents. We report a strong modulation in the conductivity and optical transmission with infrared radiation and temperature. These results are very promising for vanadium oxide based uncooled-IR sensors and smart windows that can be grown at low temperatures using a commercially viable cathodic vacuum arc-deposition technique.In conclusion, we provide a detailed in-depth understanding of electronic and optical properties of VThere are no conflicts to declare.RA-013-D3RA00752A-s001


Energy transfer via antenna effect clearly reveals the effective transfer of energy from the chromophoric moiety to the Ln3+ ion. The prepared conducting and luminescent Ln(iii) complexes might be employed as the emitting component in designing OLEDs.A series of lanthanide complexes have been synthesized with fluorinated 1,3-diketones and heteroaromatic ancillary moieties. Spectroscopic studies reveal the attachment of the respective lanthanide ion to the oxygen site of β-diketone and nitrogen site of auxiliary moieties. The conducting behavior of the complexes is proposed by their optical energy gaps which lie in the range of semiconductors. The emission profiles of the lanthanide complexes demonstrate red and green luminescence owing to the distinctive transitions of Sm A series of lanthanide complexes have been synthesized with fluorinated 1,3-diketones and heteroaromatic ancillary moieties. The above outcomes demonstrate the weak and low luminescent efficiency of lanthanides.12,13 Thus, trivalent ions typically form coordinated complexes with organic ligands. These organic moieties possess strong absorption in the UV-vis region and upon coordination with the metal ion transfer the absorbed energy to it. Therefore the organic moiety proficiently sensitizes and improves the photoluminescence efficiency of 4f ions. This mechanism is titled as “antenna effect”.14–16 Existence of solvent units in lanthanide complexes are greatly unsuitable on account of highly energized stretching vibrators of hydroxyl and amino modes which leads to luminescence quenching.17,18 It can prominently restrict the practicability of ternary complexes as luminescent material. The high energetic oscillators in chromophoric moieties are also reasonable to quench the luminescence phenomenon in lanthanide ions.19,20 Thus, the substitution of C–H by less energetic C–F bond minimizes the luminescence quenching. The additional assistance of fluorination is the heavy atom effect, which can increase the efficiency of intersystem crossing (ISC) from singlet to triplet level of ligand.21–23 Neutral ligands can substitute the solvent molecules and form coordinatively saturated lanthanide complexes via hard donor sites i.e. nitrogen and oxygen. These ligands have provided rigidity and thermal stability to the complexes. Asymmetric coordinating environment around central ion has also enhanced the luminescence characteristics.24The coordination compounds of lanthanide (Ln) ions having 3+ and Sm3+ ions for synthesis of ternary complexes. Tb3+ ion exhibits green emission which is the component of red-green-blue system accredited to transition of 5D4 → 7F5 and situated about emissive wavelength of 545 nm.25,26 The ternary samarium complexes show red luminescence due to 4G5/2 → 6H9/2 (648 nm) transition.27,28Lanthanide ions such as europium, samarium and terbium have exhibit bright red, orange and green emission, respectively in visible-range of the electro-magnetic spectrum (EMS). Now we have taken Tbiii) complexes based on fluorinated di-ketone 2,2-dimethyl-6,6,7,7,8,8,8-heptafluoro-3,5-octanedione (Hfodo) and N-donor ancillary units which are 2,2′-bipyridine [Bpy], 5,5′-dibromo-2,2′-bipyridine [DBr], 5-bromo-5′-thien-2-yl)-2,2′-bipyridine [MD] and 5,5′-bisthien-2-yl)-2,2′-bipyridine [DD]. The main aim of our work is to assess the electronic impact of substituents on the optoelectronic and photophysical characteristics of synthesized complexes. The complexes Tb(Hfodo)3Bpy (T1), Tb(Hfodo)3DBr (T2), Tb(Hfodo)3MD (T3), Tb(Hfodo)3DD (T4), Sm(Hfodo)3Bpy (S1), Sm(Hfodo)3DBr (S2), Sm(Hfodo)3MD (S3) and Sm(Hfodo)3DD (S4) have been reported. These lanthanide complexes were examined by elemental investigation, spectroscopically, thermal gravimetric and electro-chemical technique. Complexes were colorimetrically analyzed by using the results of photoluminescence emission.Here, we have reported eight Ln(22.13·6H2O and SmCl3·6H2O were bought from Sigma Aldrich (SA). The reagent such as 25% NH4OH solution and solvents viz., methyl carbinol and hexyl hydride has been utilized directly. The ligands namely DBr, MD and DD have been synthesized in lab.29 The composition of CHN in lanthanide complexes was inspected via 2400-CHN Analyzer. The FTIR and proton NMR spectral data was measured on a PerkinElmer 400 FTIR spectrophotometer and FT NMR spectrometer respectively. The proton NMR signals were obtained in CDCl3 and [(CH3)4Si] as reference. Absorption and electrochemical analyses were performed on as respective instrument such as Shimadzu UV VIS 2450 and Potentiostat-4000. PL spectral information was measured on a Horiba Fluorolog 3. Thermal-gravimetric [TG] and differential-thermal-gravimetric [DTG] patterns were obtained under nitrogen atmosphere on a Hitachi Simultaneous Thermogravimetric Analyzer-7300.The chemicals comprising Bpy, Hfodo, TbCl2.230–33 Ln(iii) complexes were made by adding 6.48 mmol of 25% NH4OH to 6.48 mmol of Hfodo in 5 mL of methyl carbinol. Beaker was retained undisturbed and closed till the smell of ammonia evaporates. Then, 2.16 mmol of alcoholic solution of lanthanide (respective samarium and terbium) chloride hexahydrate and substituted 2,2′-bipyridine ligands were dispensed to ammoniated solution of Hfodo. The resulting mixture (pH = 6–7) was agitated for 12 hours. After that, leaves the beaker for slow disappearance of solvent. Methyl carbinol and hexyl hydride (2–3 times) were employed to wash the solid residue.33.1The basic (CHN) compositional data as well as preliminary findings such as appearance and quantity of the complexes is listed in 3.2iii) complexes are mentioned in −1 indicates that water molecules do not exist in the complexes. Stretching vibrations of  PBM data was replaced with SVG by xgml2pxml:<glyph-data id="z.dbd" format="PBM" resolution="300" x-size="8" y-size="10" xml:space="preserve">00000000000000000000000000000000111111110000000011111111000000000000000000000000</glyph-data><svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="13.200000pt" height="16.000000pt" viewBox="0 0 13.200000 16.000000" preserveAspectRatio="xMidYMid meet"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform="translate scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>C–H unit are liable for the peak at 3060 cm−1. The FTIR spectrum of diketone ligand reveal a prominent peak due to CO group which is moved to the lesser frequency after complexation. This suggests the attachment of the diketone to metal ion via oxygen.34,35 The finding of peaks at about 1070–1100 cm−1 correspondent to C–Br unit existed in T2, T3, S2 and S3. In FTIR profiles of some complexes  two peaks in scope of 688–722 cm−1 are present due to C–S–C bond.29 The peaks around 455–475 cm−1 (Ln–O) and 531–538 cm−1 (Ln–N), confirms the involvement of Hfodo and ancillary moieties with the Ln3+ ion.36–38FTIR spectral information of Ln signal of Hfodo is displaced towards the lower chemical shift. The paramagnetic property of terbium and samarium ions causes noticeable alterations in the 1H-NMR spectrum data of ligands.39–41 The paramagnetic nature of Tb3+ greatly affects the position of signals of auxiliary units. Fig. S1–S163.3iii) complexes were recorded in dichloromethane (DCM) solvent (10−5 M). iii) complexes. The spectral profiles of synthesized complexes specify a band in 280–380 nm associated with the π–π* transitions of chelated moieties. Bathochromic shift is observed with change in ancillary moieties from Bpy to DD. Bands due to free units get displaced after complexation with metal ion.42,43 With the assistance of spectral profiles, optical band-gap (Eg) was designed ; energy of photon (ℏν) and optical parameter (n).44,45Eg was determined by extrapolating a line to (αhυ)2 = 0 as given in Eg declines in a particular series i.e. from T1 to T4 and S1–S4 suggesting the increase in conjugation. The band gap value lies in the range of semiconducting materials.The above relation includes absorption co-efficient (3.4iii) complexes were collected in order to measure their thermal strength. The thermograms of T1, T4, S1 and S4 are thoroughly examined here because the disintegration tendencies of all the prepared complexes are quite comparable. iii) ion. In case of S1 and S4, huge mass loss of 89.67%  and 90.15%  in 225–343 °C and 246–366 °C is attributed to removal of three Hfodo and single neutral unit respectively. The residual product formed after decomposition was due to oxides of terbium and samarium.46–48 The peaks in DTG curves at 267 °C (T1), 275 °C (T4), 308 °C (S1) and 358 °C (S4) support their decomposition pattern.The thermograms of Ln(3.5−1 are often used to record cyclovoltammograms (CV). 0.1 M tetrabutylammonium perchlorate in DCM was used as supporting electrolyte and silver wire as a reference electrode. The concentration of ferrocene and the complexes were 10−3 M. CV of synthesized complexes  with ferrocene in inset is shown in 49,50A potential range of −2 V to +4 V and a scanning frequency of 0.1 V s5129,52Ferrocene (Fc) has a half potential of 1.03 V.3.6θ in range of 10°–50°. The sharp peaks in XRD profiles suggest that the crystalline nature of synthesized complexes. From powder XRD patterns, it can be found that the prepared complexes hold different degree of crystallinity.53 High crystallinity degree in ternary Ln(iii) complexes is evidenced by better defined peaks in their diffractogramms.To get an idea about crystalline nature of synthesized ternary complexes, their powder X-ray diffraction (XRD) patterns were recorded. 3.7iii) complexes evince the broad band of ligand as well as few weak intense peaks owing to f–f transitions of metal ion. Emission spectral profile of ternary terbium complexes were obtained at their respective excitation wavelength (λex) in solid state. Emission spectra display peaks in 480–620 nm, characteristic of Tb3+ ion. The peaks in photoluminescence emission spectra are positioned at 490, 546, 590 and 617 nm attributed to transitions from excited 5D4 state to lower situated 7F6, 5, 4, 3 states of Tb3+ ion separately.54,55 Along with these peaks, the least intense peaks were also seen corresponding to 5D4 → 7F2–0. Dominant peak present at around 546 nm is answerable for green emission of T1–T4 complexes.56The photoluminescence profiles of terbium (T1–T4) and samarium (S1–S4) complexes monitored in solid form are demonstrated in f–f transitions of Sm3+ ion. Emission profiles show characteristic peaks of at 564 nm (04G5/2 → 06H5/2), 601 nm (4G 5/2 → 6H7/2), 648 nm (4G5/2 → 6H9/2) and 707 nm (4G5/2 → 6H11/2) transition.57,58 Ligand dependent bands were appeared in emission spectra of S3 and S4. The emission spectra of S1–S4 evident most pronounced peak at 648 nm depends on the coordinating surrounding of Sm3+ ion.59 The PL emission intensity decreases in a particular series however it is larger than their binary complex. Hence, the prepared complexes showed enhanced luminescence behavior. The order of emission intensity in synthesized complexes is also supported by energy transfer phenomenon as shown in iii) i.e. [Gd(Hfodo)3(H2O)2]. The shortest wavelength in the phosphorescence spectrum provides the energy of triplet state of Hfodo. The T level energy of Bpy and its derivatives has already been reported in literature.60 Energy transfer parameters of diketone and neutral moieties are listed in Table S1.†β) of synthesized complexes.61AψJ–ψ’J denotes the integrated area of PL emission peaks. The value of β for hyper-sensitive peak is ∼60% as listed in Tables S262 The intensity ratio of 5D4 → 7F6/5D4 → 7F5 for Tb(iii) and 4G5/2 → 6H9/2/4G5/2 → 6H5/2 for Sm(iii) demonstrates the asymmetric environment around metal ion.63 The quantum yield for synthesized Ln complexes is measured against the reference i.e., quinine bisulphate in dilute sulphuric acid. It is calculated in order to investigate the influence of different neutral moieties on the PL intensity of complexes. Φs).64Φr, A, I, s, r and n represent quantum yield reference, absorbance at the excitation wavelength, integrated emission intensity, sample, reference and refractive index of solvent, respectively.65 The value of Φr is 54.6%. Quantum yield is found to be in the declining manner and support the outcomes obtained from emission spectral profiles. The numerous absorption and photo-physical data are compiled in Excitation spectra of S1–S4 evince broad band of ligand as well as few weak intense pinnacles relative to 3.8x, y) co-ordinates was obtained from emission spectral data. These co-ordinates positioned in the greenish and orange red regime of x, y) diagram of complexes. From , the other colorimetric parameters i.e.,  and color temperature (CT) were estimated through 66–68  with CT values are manifested in The color (n characterizes the inverse-slope line and was assessed viaxe and ye represents the color epicenter having respective entity i.e. 0.332 and 0.186. Color purity (CP) of prepared Ln complexes was determined from xi & yi) with value 0.333 are the illuminated points. The value of dominant points xd = 0.290 (green) & 0.688 (red) and yd = 0.600 (green) & 0.331 (red).69–71 The relative luminance value for a certain color in terms of RGB parameters is also calculated. The color characteristics for ternary lanthanide complexes are listed in In the relation 4β value corresponding to the hyper-sensitive transition and the range of band gap further prove their utility as conducting material in fabricating displays.A different series of Ln complexes with fluorinated primary and heteroaromatic secondary ligand have been prepared and examined. The outcome of infrared study reveals the coordination of ligands through hard donor atoms (–O and –N). The complexes exhibit band at higher wavelength as compared to Hfodo which illustrates the stability of ligand orbitals on complexation. The high luminescent intensity recommends the superior ligand sensitization for solid sample. Synthesized terbium and samarium complexes emit bright green and orange red emission which is the constituent of tricolor system. The prepared lanthanide complexes are potential applicant in laser diodes due to high The article does not involve any study performed on animals or human by any of the authors.The authors affirm that the information/data of this research article is available inside the article.Anjli Hooda = data curation, writing – original draft; Devender Singh = writing – review & editing, supervision; Anuj Dalal = investigation; Kapeesha Nehra = formal analysis; Sumit Kumar = visualization; Rajender Singh Malik = software; Ramesh Kumar = methodology; Parvin Kumar = resources; Brijesh Rathi = validation.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.RA-013-D3RA00214D-s001