The Bacillus subtilis endo-β-1,4-glucanase (BsCel5A) hydrolyzes β-1,3-1,4-linked glucan, and the enzyme includes a family 3 carbohydrate-binding module (CBM3) that binds β-1,4-linked glucan.Here we investigate the BsCel5A β-1,3-1,4 glucanase activity after exchanging the CBM3 domain for the family 11 CBM from Ruminiclostridium thermocellum celH (RtCBM11) having β-1,3-1,4 glucan affinity.The BsCel5A-RtCBM11 presents a 50.4% increase in Vmax, a 10% reduction in K0.5, and a 2.1-fold increase in catalytic efficiency. Enzyme mobility and binding to barley β-1,3-1,4 glucan and pre-treated sugarcane bagasse were investigated using Electron Paramagnetic Resonance (EPR) with Site-Directed Spin Labeling (SDSL) of the binding site regions of the CBM3 and RtCBM11 domains in the BsCel5A-CBM3 and BsCel5A-RtCBM11, respectively. Although higher mobility than the RtCBM11 was shown, no interaction of the spin-labeled CBM3 with β-1,3-1,4 glucan was observed. In contrast, a Ka value of 0.22 mg/mL was estimated from titration of the BsCel5A-RtCBM11 with β-1,3-1,4 glucan. Enzyme binding as inferred from altered EPR spectra of the BsCel5A-RtCBM11 was observed only after xylan or lignin extraction from sugarcane bagasse. Binding to xylan- or lignin-free lignocellulose was correlated with a 4.5- to 5-fold increase in total reducing sugar release as compared to the milled intact sugarcane bagasse, suggesting that xylan impedes enzyme access to the β-1,3-1,4 glucan.These results show that the non-specific binding of the BsCel5A-RtCBM11 to the lignin component of the cell wall is minimal, and represent the first reported use of EPR to directly study the interaction of glycoside hydrolyse enzymes with natural insoluble substrates.The online version of this article (10.1186/s13068-017-0964-0) contains supplementary material, which is available to authorized users. Cellulose is the major component of the plant cell wall, and is integrated with various other polysaccharides and lignin to form a complex polymer matrix. Cellulose forms linear chains comprising glucose monomers linked by β-1,4-glycosidic bonds . The enzhttp://www.cazy.org) that display variable binding specificities against different plant cell wall polysaccharides such as crystalline or amorphous cellulose, β-glucans, xylans, laminarins, mannans, galactans, xyloglucans, arabinans, and others.Endo- and exoglucanases frequently comprise two or more domains that display multi-functional properties, where a catalytic domain (CD) is responsible for the hydrolytic activity, and non-catalytic domains enhance catalysis or perform structural functions . The nonin natura methyl]-1H-pyrrol-1-yloxy (MTSSL) for 24 h at 25 °C. The modified protein was separated from excess free spin label with a Superdex 200 column (5 × 150 mm) eluted with 40 mM HEPES, 300 mM NaCl, and pH 8.0 buffer. The spin-labeled proteins were concentrated to 200 μM by ultrafiltration.Ms = (δexp−1 − δi−1)/(δm−1 − δi−1), where δexp is the value of the central line width of R1 in the MTSSL-labeled protein. The δi = 8.4 G and δm = 2.1 G are the values of the greatest degree of either immobilization or mobility of sites observed in a protein, respectively, as obtained in studies with rhodopsin [Continuous wave EPR spectroscopy was performed at room temperature using a JEOL JES-FA200 equipped with temperature control and a cylindrical cavity operating in X band mode. Spectral analyses were performed according to empirical parameters as previously described . The Ms mobility parameter was calculated as function of the central line width as hodopsin , 18.Ka).Solutions containing labeled proteins free in solution, and in the presence of either purified β-glucan or pre-treated sugarcane cell wall samples were prepared and drawn into capillary tubes. The final protein concentration was 100 μM, and the β-glucan concentration was varied from 0.1 to 5 mg/mL. The sugarcane cell wall preparations were used at a concentration of 1 mg/mL. The titration curve was fitted to a sigmoidal Hill function by non-linear regression using the program Microcal Origin version 8.0, to yield estimates of the association constant  ethanol at 80 °C for 20 min to remove sugars and other soluble compounds. The alcohol insoluble residue (AIR) was washed with distilled water and dried at 60 °C for 24 h . The AIRfor 24 h . Subsequfor 24 h , where afor 24 h . Biomassfor 24 h  where apfor 24 h , 42.The activity of the enzymes against AIR and all the treated lignocellulosic substrates was performed using a 1% w/v suspension of the treated substrate prepared in 50 mM Bis–Tris buffer, pH 6.0, buffer, 100 μM of either BsCel5A-CBM3 or BsCel5A-RtCBM11 at 50 °C for 24 h in an orbital shaker at 200 rpm. The release of total reducing sugars was measured by the DNS method .Bscel5A-CBM3 and Bscel5A-RtCBM11 nucleotide coding sequences encode polypeptides of 494 and 514 amino acids, respectively, and were successfully expressed as soluble proteins in E. coli Roseta (DE3). The molecular masses of the cysteine mutant proteins BsCel5A-CBM3 and BsCel5A-RtCBM11 were 54,787.9 and 56,897.3 Da, respectively, including the polyhistidine sequence added to the N-termini derived from the pET28a plasmid. The relative molecular mass of both enzymes estimated by gel filtration was approximately 55 kDa (data not shown), and it was therefore assumed that both enzymes are monomeric. The far-UV CD spectra of the pure enzymes show a maximum at approximately 195 nm, a minimum around 215 nm, and a shoulder at 225 nm  of the BsCel5A catalytic domain when the CBM3 was replaced by RtCBM11. The catalytic efficiency (kcat/K0.5) of β-glucan hydrolysis was 53% greater for BsCel5A-RtCBM11 as compared to BsCel5A-CBM3. Both enzymes presented similar K0.5 values for CMC hydrolysis, suggesting that the catalytic domain can maintain hydrolytic function independent of the associated CBM. This result is in agreement with the previously known affinities of the CBMs for the polysaccharide used in this study, where RtCBM11 shows a higher affinity for β-glucan as compared to CBM3, and neither CBM3 nor RtCBM11 presents significant binding to CMC [The endoglucanase activity with different concentrations of either β-glucan or CMC substrates see Fig.  was highg to CMC .Fig. 1ThExperimental strategies used to study polysaccharide binding to CBMs include affinity electrophoresis, insoluble polysaccharide pulldown, and NMR . Here weendo-binding) in a cleft on the protein structure, whereas Type C CBMs have shortened pockets that bind to the termini of the polysaccharides (exo-binding) [Bacillus subtilis CBM3 (BsCBM3) and the Type B Ruminiclostridium thermocellum CBM11 (RtCBM11) are presented in Fig. Type A CBMs interact with the smooth surface of crystalline cellulose with a planar hydrophobic carbohydrate-binding site , 12, Typbinding) . The cryY151R1 and BsCel5A-CBM3C405R1 and used in SLDL-EPR experiments.The CBM3 domain from BsCel5A and the RtCBM11 structures presented in Fig. Y151R1 and BsCel5A-CBM3C405R1 in the presence and absence of β-glucan. All spectra show three prominent narrow lines typical of nitroxide spin probes  of 0.23 ± 0.01 mg/mL. This value is consistent with the previously reported Ka value of 0.24 mg/mL for cooperative binding of the RtCBM11 protein to β-glucan as estimated from isothermal titration calorimetry experiments [The titration of BsCel5A-RtCBM11opy Fig. a. An incopy Fig. b. The ve1R1 Fig. b, and reeriments .Fig. 4aexo-binding at the polysaccharide terminus generated by hydrolysis favors dissociation even at high β-glucan concentrations, thereby liberating the enzyme for high-affinity endo-binding to the substrate. We note that no significant change was observed in the K0.5 between the BsCel5A-CBM3 and BsCel5A-RtCBM11 constructs, further indicating that the catalytic and carbohydrate-binding domains can function independently. It has previously been observed that a xyloglucan-specific endo-β-1,4-glucanase from Aspergillus niveus (XegA) fused to a xyloglucan-specific CBM44 domain from the CelJ of R. thermocellum (RtCBM44) exhibits a 30% increase in the catalytic efficiency for xyloglucan as compared to the parental enzyme [Comparison of the titration results from EPR with the results from reducing sugar release reveals that the RtCBM11 binding is saturated at β-glucan concentrations of less than 0.5 mg/mL, whereas the measured catalytic activity of the BsCel5A-RtCBM11 continues to increase even at a β-glucan concentration of 15 mg/mL. The high-affinity association of the Type B RtCBM11 within the β-glucan might be expected to facilitate the binding of the BsCel5-RtCBM11 and enhance catalysis by the active site domain. After hydrolysis, we propose that a reduced affinity of the RtCBM11 l enzyme . It was l enzyme .Y151R1 in the presence of untreated milled sugarcane bagasse . The EPR spectra of the BsCel5A-RtCBM11drolysis , 55. Thedrolysis , 56. TheO-4a alkyl-ari-ether bonds [Y151R1 in the presence of the delignificated cell wall fraction (Fig. The final fractionation step is the removal of lignin, which has a supramolecular polymeric structure derived from aromatic groups linked by β-er bonds , 58. Theer bonds , and it er bonds –63. The Y151R1 binding observed in the EPR experiments. It has been demonstrated that non-selective binding of hydrolytic enzymes to plant cell wall lignin can reduce their catalytic activity [Y151R1 to treated sugarcane by direct measurement with EPR spectroscopy. In contrast to many previous reports, our results have demonstrated that only low levels of protein bind to samples in which lignin is present. Our results further indicate that in the case of the BsCel5A-RtCBM11, removal of the O-acetyl-4-O-methylglucurono-xylan with DMSO is the key treatment of the sugarcane resulting in both an increase in enzyme binding and a concomitant increase in reducing sugar release. The protective effect of the O-acetyl-4-O-methylglucurono-xylan suggests that the sequence of deconstruction of sugarcane cell wall requires the prior enzymatic or chemical removal of xylans for the exposure to hydrolysis by the β-glucanase. This implies that β-glucanase activity against cell wall lignocellulose would be enhanced in the presence of a xylanase, and indeed synergy between these two enzymes has been observed in the hydrolysis of wheat lignocellulose [These results were correlated with the results of total reducing sugar release using sugarcane bagasse after the same sequence of treatments as used in the EPR experiments. Reducing sugar release Fig. b on incuactivity , and it activity . The majellulose .Here we have shown that substitution of the naturally occurring CBM3 domain in the BsCel5-CBM3 by the RtCBM11 results in an increase in the hydrolytic efficiency of the enzyme. We have also shown for the first time through direct measurements by EPR spectroscopy that the CBM3 of the BsCel5A-CBM3 does not bind to β-glucan substrate, suggesting that the CBM3 and catalytic domains may act independently. The EPR measurements further reveal that the RtCBM11 of the BsCel5A-RtCBM11 displays high affinity for β-glucan, which is correlated with the increase in catalytic efficiency of the enzyme against this polysaccharide. To fully exploit the potential of EPR spectroscopy for the study of CBM binding in a biologically relevant environment, we have analyzed the association of the BsCel5A-RtCBM11 against a series of insoluble sugarcane bagasse fractions. The results suggest that hemicellulose impedes the binding of BsCel5A-RtCBM11 and the hydrolysis of the β-glucan in the sugarcane cell wall. This demonstrates the potential of EPR spectroscopy to study the interaction of hydrolases with complex insoluble lignocellulosic materials.


Naringin, a plant bioflavonoid, has anti-inflammatory and anti-oxidative properties. We hypothesized that P2X7 receptor mediated gp120-induced injury in primary cultured microglia, and that naringin would have a protective effect. We showed that HIV-1 gp120 peptide  appeared to induce apoptosis of primary cultured microglia. However, there was a decrease of microglia apoptosis in gp120+naringin group compared with gp120 group. Using qPCR, Western blot, and immunofluorescence, we showed that gp120 stimulated expression of P2X7 mRNA and receptor protein, and this stimulation was inhibited by naringin. Treatment with gp120 increased concentrations of eATP, TNFα and IL-1β, and these effects were inhibited by naringin. Taken together, these results suggested that gp120 contributed to microglial cell injury and neurotoxic activity by up-regulating expression of P2X7, in a naringin-protective manner.Human immunodeficiency virus type-1 (HIV-1) envelope glycoprotein 120 has been shown to activate microglia, causing release of inflammatory and toxic factors. The P2X As the efficacy of antiretroviral therapy (ART) increases, the short-term mortality of acquired immune deficiency syndrome (AIDS) has decreased somewhat. AIDS is becoming a chronic disease with increased longevity . The ext7 receptor, a purinergic receptor primarily expressed on macrophages and microglia, is associated with the release of inflammatory factors [7 receptor by extracellular ATP (eATP), released from injured cells, activates microglia and results in the release of inflammatory cytokines that are implicated in the pathogenesis of many neuronal disorders [7 receptor may represent a new target for the prevention of microglia injury and the treatment of ADC and other neuronal disorders associated with the inflammatory cascade.P2X factors . Activatisorders –12. Actiisorders . Taken tNaringin is a type of plant bioflavonoid extracted mainly from grapefruit and other related citrus species. It possesses various biological and pharmacological properties including anti-inflammatory, anti-oxidative and anti-apoptotic properties ,15. In a7 receptor is closely related to inflammation, and that naringin exerts protective effect against inflammation and apoptosis, we hypothesize that the anti-inflammatory property of naringin may be related to inhibition of the P2X7 receptor. In this study, we explore the role of the P2X7 receptor in the pathogenesis of microglia injury, and we investigate the potential protective effect of naringin on gp120-induced primary cultured microglia injury.Based on the fact that P2X2 flasks which had been coated with poly-l-lysine (PLL). Each flask contained 6 ml DMEM/F12 medium  which included 10% FBS  and 1% PEN/Strep . Cell concentration was approximately 106 cells/ml calculated by blood cell counting chamber. The cells were cultured in a constant-temperature incubator  containing 5% CO2 at 37°C. After having been cultured for 7–9 days, microglia were isolated from the mixed glia by gentle shaking [5 cells/ml. After 30 min, culture medium was changed to remove non-adhering cells.This study was approved by the Basic Medical College of Nanchang University. Sprague-Dawley rats (1–2 days old) were obtained from the Laboratory Animal Science Department of Nanchang University. Animals were treated according to the guidelines for Care and Use of Animals set by the Ethical Committee of Nanchang University. Neonatal rats were anesthetized hypothermically using ether. Then they were fully disinfected with 75% alcohol under anesthesia, and decapitated while being placed on ice. Whole brain was dissected to obtain mixed glia cells. The meninges and blood vessels were removed carefully using ophthalmic forceps. Brain tissues were then minced using ophthalmic scissors, and enzymatically digested using 0.125% trypsin at 37°C for 60 min. Samples were separated by centrifugation at 1000 rpm for 10 min and filtered through a 70 μm cell strainer. The mixed cells were cultured on 25 cm shaking . HarvestIn subsequent experiments, purified microglia were divided into four groups at random: control group (Ctrl); gp120 model group (gp120); naringin treatment group (gp120+naringin); and drug solvent control group (gp120+DMSO). After microglia were inoculated into plates, all groups apart from Ctrl group were incubated with 2.0 μg/L gp120 for 24 h. Gp120+naringin group was treated with 80 μM naringin (dissolved in DMSO), and gp120+DMSO group was treated with 0.08% DMSO. The concentration of naringin  that we used was based on our previous study . Naringi5 cells/ml in 24-well plates. After drug treatment for 24 h, microglia were washed three times in PBS and fixed with 4% paraformaldehyde (PFA) for 15 min at room temperature. After washing, microglia were permeabilized in 0.3% Triton X-100 for 10 min and blocked with 10% normal goat serum for 1 h. Cells were then incubated with CD11b primary antibody  overnight at 4°C. After washing in PBS, microglia were incubated with FITC secondary antibody  for 1 h. Cells were then incubated with DAPI  for 90 s for nuclear staining. Finally, preparations were mounted on slides and imaged with a fluorescence microscope .Immunofluorescence was performed to identify the purity of primary cultured microglia. Purified microglia were inoculated on coverslips that had been coated with PLL at a density of 10TUNEL assay was performed to detect cell apoptosis using the In Situ Cell Apoptosis Detection Kit . Microglia were inoculated on coverslips that had been coated with PLL and treated with reagents. After 24 h, microglia were washed three times in PBS and fixed with 4% PFA for 30 min at 37°C. After washing three times with PBS, microglia were digested using Proteinase K (1:200 diluted in 0.01M TBS) at room temperature for 30 s and washed three times in 0.01M TBS. According to the manufacturer’s instructions, microglia were incubated with TUNEL reaction reagent, biotin-labeled anti-digoxin antibody, and SABC diluent. Microglia were then incubated with DAPI for nuclear staining, and finally mounted to the slides. Apoptotic cells were imaged by a fluorescence microscope.5 cells/ml per well. Each group was set with three parallel wells. After reagent treatment had been applied for 24 h, the culture medium of each group was harvested to detect levels of eATP with ATPlite one step kit . Lyophilized substrate solution was reconstituted using the appropriate volume of buffer. A vial of lyophilized ATP standard solution was reconstituted with water, and ATP standard solution was diluted into 1000, 500, 250, 125, 62.5, 31.2, 15.6, 7.8 pM for obtaining standard curve. For detection, we used 384-well opaque plates, with each well containing either 25 μl sample or ATP standard. Another 25 μl substrate solution was added to each well. After that, the 384-well microplate was shook for two minutes at 1,100 rpm. A multifunctional microplate reader  was used to measure the luminescence of each group. The ATP content of each group was calculated using a standard curve according to manufacturer’s instructions. The DOI link of our protocol is http://dx.doi.org/10.17504/protocols.io.i9tch6n.After isolation and purification, microglia were seeded on 24-well plates at a density of approximately 107 mRNA. After extraction, total RNA was quantified by measuring OD value at 260 nm, followed by reverse transcription using 25 ng of total RNA. Each sample included an RNA specific control for the evaluation of potential contamination of genomic DNA. We used a SYBR® Green detection system to quantify expression of P2X7 at mRNA level using the ABI PRISM® 7500 Sequence Detection System . PCR amplification was performed using 2 μl cDNA, with a total volume of 20 μl. Each sample was assayed in triplicate. The upstream and downstream primer sequences of the P2X7 were as follows: 5′-TTACGGCACCATCAAGTGGA-3′ and 5′-GCAAAGGGAGGGTGTAGTCG-3′, respectively, with the expected product size being 218 bp. The expression level of β-actin was chosen as a reference. Forward and reverse primer sequences  of β-actin were used for amplification, and the expected product size was 110 bp. Cycling parameters were as follows: 94°C for 30 s to activate DNA polymerase; 40 cycles of 94°C for 5 s and 60°C for 30 s for amplification; 95°C for 15 s, 60°C for 1 min, and 95°C for 15 s to obtain the melt curve. The average threshold cycle (CT) value of P2X7 minus the average CT value of β-actin was the ΔCT value (ΔCT = CT target—CT reference). Results were calculated as follows: ΔΔCT = ΔCT test sample—ΔCT calibrator sample. Expression levels of P2X7 (RQ) were calculated using the following equation: RQ = 2-ΔΔCT.Quantitative real-time PCR (qPCR) was performed to measure the expression of P2X7 , and β-actin . The secondary antibody was goat anti-rabbit/mouse IgG (Beijing Zhongshan Biotech Co.). Band densities were normalized with β-actin internal controls.After reagent treatment for 24 h, primary cultured microglia seeded on 6-well plates were lysed in RIPA buffer  for protein extraction. The composition of RIPA buffer was as follows: 50 mM Tris (pH 7.4), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, sodium orthovanadate, sodium fluoride, EDTA and leupeptin. The lysate was centrifuged at 12000 rpm for 5 min at 4°C. The supernatant was collected and preserved at -20°C for later use. The protein content of the samples was determined via the Lowry method. Samples containing equal amounts of protein (20 μg) were diluted with sample buffer, heated to 95°C for 10 min, and separated on SDS-polyacrylamide gels (10%) using an electrophoresis device . Proteins were transferred onto PVDF membranes. A multifunctional gel imaging system was used to detect chemiluminescent signals. Band density was quantified using Image-Pro Plus 6.0 software . The primary antibodies were as follows: rabbit polyclonal anti-P2X7 and CD11b in primary cultured microglia. Primary antibodies were rat anti-CD11b primary antibody  and rabbit anti-P2X7 antibody . Secondary antibodies were goat anti-rabbit TRITC IgG and goat anti-mouse FITC IgG . Other experimental steps were performed as described in the section of primary cultured microglia purity identification.Immunofluorescence was performed to identify co-expression of P2XAfter drug treatment for 24 h, the culture medium of each group was harvested to detect levels of TNFα and IL-1β via ELISA . A multifunctional microplate reader  was used to measure the OD values of each group. TNFα and IL-1β contents were calculated using a standard curve according to manufacturer’s instructions. The experiment was performed as described previously .Fisher’s Least Significant Difference (LSD) test was used for multiple comparisons. Significance was defined as P < 0.05.SPSS 21.0  was used to analyze the data. All results were expressed as mean ± SD. Statistical significance was determined by one-way analysis of variance (ANOVA), and Prior to the performance of subsequent experiments, immunofluorescence was used to identify the purification degree of isolated primary microglia using the specific marker CD11b. By immunofluorescence, we showed that the purity of primary cultured microglia was greater than 90%. This level fully conformed to the requirements of subsequent experiments .7 receptor expression in microglia, qPCR and Western blot were used to detect the mRNA and protein expression of P2X7. Compared with Ctrl group, expression of P2X7 mRNA and receptor protein in gp120 group was significantly up-regulated (P < 0.01), suggesting that gp120 up-regulated expression of P2X7 receptor in primary cultured microglia . Compared with Ctrl group, apoptotic primary cultured microglia in gp120 group increased significantly (P < 0.01), suggesting that gp120 induced apoptosis of microglia. There was no apparent difference in microglia apoptosis between gp120 and gp120+DMSO groups (P > 0.05), suggesting that DMSO had no significant effect on primary cultured microglia apoptosis. There was a decrease in cell apoptosis in gp120+naringin group compared with gp120 group (P < 0.01), suggesting that naringin had an inhibitory effect on gp120-induced apoptosis in microglia  in Ctrl, gp120, gp120+naringin and gp120+DMSO groups was 7.15 ± 2.10, 36.81 ± 4.66, 19.37 ± 3.71 and 40.17 ± 5.20, respectively. Differences among groups were statistically significant  of Ctrl, gp120, gp120+naringin and gp120+DMSO groups were 19.39 ± 6.25, 99.89 ± 17.86, 40.30 ± 11.66 and 94.73 ± 13.93, respectively. Differences among groups were significant . The contents of eATP in gp120 and gp120+DMSO groups were significantly higher than that of Ctrl group (P < 0.01). The concentration of eATP in gp120+naringin group was significantly lower than gp120 group (P < 0.01). There was no statistical significance between gp120+naringin and Ctrl groups with respect to ATP concentration (P > 0.05), suggesting that naringin may inhibit excessive release of ATP by injured cells, thus exerting a protective effect on gp120-induced primary cultured microglia injury . Expression of P2X7 mRNA in gp120 group was significantly higher than that of Ctrl group (P < 0.01), suggesting that gp120 may promote up-regulation of P2X7 mRNA. There was no significant difference in P2X7 mRNA expression between gp120 and gp120+DMSO groups (P > 0.05). This suggested that DMSO had no significant effect on P2X7 mRNA expression in primary cultured microglia. Expression of P2X7 mRNA in gp120+naringin group was substantially lower than that of gp120 group (P < 0.05), suggesting that naringin had an inhibitory effect on P2X7 mRNA expression . Expression of P2X7 receptor protein in gp120 group was significantly higher than that of Ctrl group (P < 0.01), suggesting that gp120 up-regulated expression of P2X7 receptor protein. There was no significant difference in P2X7 protein expression between gp120 and gp120+DMSO groups, suggesting that DMSO had no significant effect on P2X7 protein expression. Expression of P2X7 receptor protein in gp120+naringin group was much lower than that of gp120 group (P < 0.01), suggesting that naringin inhibited gp120-induced up-regulation of P2X7 receptor protein expression in primary cultured microglia . Compared with Ctrl group, co-expression levels of CD11b and P2X7 receptor in gp120 and gp120+DMSO groups were higher (P < 0.01), while co-expression values of CD11b and P2X7 in naringin+gp120 group were markedly lower than those of gp120 group (P < 0.01). This suggested that naringin reduced expression of P2X7 receptor in primary cultured microglia induced by gp120 , while there was no statistical difference between gp120 group and gp120+DMSO group (P > 0.05). This suggested that gp120 induced release of TNFα and IL-1β in primary cultured microglia. There was no statistical significance between gp120+naringin and Ctrl groups with respect to TNFα concentration (P > 0.05). Levels of TNFα and IL-1β in gp120+naringin group were lower than those of gp120 group, suggesting that naringin inhibited the release of TNFα and IL-1β induced by gp120.After OD values of each group were measured with a microplate reader, TNFα and IL-1β levels of primary cultured microglia were calculated. TNFα levels (pg/ml) in Ctrl, gp120, gp120+naringin and gp120+DMSO groups were 49.81 ± 8.50, 148.97 ± 22.14, 70.64 ± 13.07 and 158.61 ± 20.70, respectively. Differences among groups were statistically significant , microglia can be activated by HIV-1 envelope glycoprotein 120 through diverse mechanisms ,22. In ampletely .7 receptor has been shown to play a crucial role in regulating the activity of microglia [7 receptors [7 receptor are exquisitely sensitive to ATP-mediated cytotoxicity. In addition, eATP induces microglia apoptosis via the activation of P2X7 receptor [7 receptor, resulting in microglial apoptosis. A possible mechanism may be that activation of P2X7 by ATP gives rise to influx of small cations, including Ca2+, Na+, and K+. This influx may lead to a series of cell-specific downstream signaling events, including formation of reactive oxygen species (ROS) and reactive nitrogen species, and either cell proliferation or cell death [P2 purinergic receptors, whose ligands are extracellular adenine nucleotides (such as ATP) are important for modulating the inflammatory functions of microglia . As a meicroglia ,28. Undeeceptors , resultieceptors . Anothereceptors . It is dreceptor ,33. Herell death . The con7 receptor, expressed at high levels on immune cells, has a more defined role than other purinergic receptors. For example, the P2X7 receptor triggers release of cytokines [7 receptors abundantly [7 mRNA and receptor protein in primary cultured microglia was significantly higher in gp120 group than Ctrl group, while expression of P2X7 mRNA and receptor protein in naringin+gp120 group was markedly lower than that of gp120 group. Considering the fact that the apoptosis rate increased considerably and the P2X7 receptor was expressed abundantly in gp120 group, we may infer that gp120 induced apoptosis of primary cultured microglia, leading to abundant ATP release, which in turn robustly activated microglia and increased expression of P2X7 receptor. We confirmed our results with immunofluorescence. We showed that co-expression of CD11b and P2X7 receptor in gp120 group was significantly higher than that of Ctrl group, and Naringin inhibited this up-regulation. Taken together, these results suggested that gp120 up-regulated P2X7 mRNA and receptor protein, on which naringin had an inhibitory effect. This may be attributed to the fact that naringin inhibited excessive activation of microglia.The P2Xytokines . Activatundantly , leadingIt is demonstrated that over-activated microglia overproduce a variety of pro-inflammatory cytokines, including IL-1β, TNFα and chemokines, all thought to give rise to ADC . One stu7 receptor. We hope that our results may facilitate the further study of HIV-1-related neurodegenerative diseases, and help improve the quality of life of those suffering from these diseases.In summary, we provide evidence that naringin exerts a partially protective effect on gp120-induced injury in primary cultured microglial cells, and the effect is most likely mediated by the P2XS1 File(XLS)Click here for additional data file.


Three positions in the ρ-Da1a evolutionary pathway, positions 28, 38 and 43 have been identified as key modulators of the affinities for the α1 and α2C adrenoceptor subtypes. Here, we present a first attempt at rational engineering of the aminergic toxins, revealing an epistasis phenomenon.Mamba venoms contain a multiplicity of three-finger fold aminergic toxins known to interact with various α-adrenergic, muscarinic and dopaminergic receptors with different pharmacological profiles. In order to generate novel functions on this structural scaffold and to avoid the daunting task of producing and screening an overwhelming number of variants generated by a classical protein engineering strategy, we accepted the challenge of resurrecting ancestral proteins, likely to have possessed functional properties. This innovative approach that exploits molecular evolution models to efficiently guide protein engineering, has allowed us to generate a small library of six ancestral toxin (AncTx) variants and associate their pharmacological profiles to key functional substitutions. Among these variants, we identified AncTx1 as the most α Recruited by convergent evolution, toxins impact mainly the haemostatic, nervous and cardiovascular systems2. Some toxins present in venoms, in spite of their debilitating effects, have become live-saving drugs5. To exert their biological activities, toxins interact mainly with ion channels, coagulation factors, nicotinic receptors, cell membrane or enzymes but only rarely with the most important class of physiological targets, i.e, the G protein-coupled receptors (GPCRs)6. Compared to numerous toxins that act on voltage-gated or ligand-gated ion channels, only a few toxins that interact with GPCRs have been identified. These toxins have been isolated mainly from cone snails venoms such as conopressin interacting with vasopressin receptor7, contulakin-G which binds to neurotensin receptor8, ρ-conotoxin TIA specific of the α1A-adrenoceptor9 and the τ-conotoxin CnVA that interacts with the somatostatin sst3 receptor10. The black widow spider and the gila monster provide further examples of GPCRs interacting toxins, namely, α-latrotoxin interacting with the latrophilin receptor11 and exenatide, exploited as an anti-diabetic drug12, that targets the GLP-1 receptor, respectively. In addition, mamba venoms contain aminergic toxins that recognize various bioaminergic receptors19.Evolutionary processes of venomous animals have selected enzymes and disulfide-rich peptides in their venoms to improve their ability to subdue their prey and defend against predators. These compounds act predominantly on few, well characterized and physiologically-relevant molecular targets of the envenomed animals20. Recently, protein engineering using a cDNA display approach was applied to 3FT to create large libraries of randomized sequences. After repeated rounds of expression and screening, interleukin-6 receptor ligands21 and serine protease inhibitors22 were selected, highlighting the high functional versatility of this structural template. On the same scaffold, a more rational protein engineering was developed using a loop grafting strategy leading to chimeric toxins with new functional profiles on muscarinic and adrenergic receptors23. Despite sequence identities that vary from 70 to 98%  superfamily, a structural fold known to support a large diversity of biological functions98% Fig. , aminerg98% Fig. . On the 98% Fig. , while o98% Fig. . These o24, fold stability over time25, protein solubility26 or to perform protein engineering. By constructing and testing function of these ancestral proteins and comparing the results with extant proteins, this method is able to follow amino acids changes in the evolutionary history and measure their impact on their properties. This “evolutionary biochemistry”27 open also a new avenue for protein engineering28 by creating small but functionally rich libraries29. As the first proof of concept for enzyme engineering, Gaucher and colleagues use the REAP (Reconstructing Evolutionary Adaptive Paths) to engineer new Taq polymerases with original properties31.In the recent years, a growing number of works relating to the use of ancestral protein resurrection have been published to address different issues like understanding protein function evolution1A and the most potent α2 adrenergic toxins.Here, we use the ancestral protein resurrection methodology on the aminergic toxin group in order to pinpoint the functional substitutions which may have occurred during their evolution and use them for molecular engineering. It is the first application of this methodology on a mini-protein/peptide family. We compare the pharmacological profiles of the natural and six ancestral toxins found at different nodes of the aminergic toxin tree and describe the functional modulations between them that could be associated with key substitutions. A first attempt to engineer more potent aminergic 3FT validates these findings by obtaining the most selective α19. Based on these results, the maximum likelihood (ML) tree of the mamba aminergic toxins was reconstructed , but only 5 and 7 substitutions spread over fingers I and II compared to ρ-Da1b and AncTx6, respectively. Finally, AncTx6 was close to MTα and MT1. Only one (I31L) and three substitutions  differentiated AncTx6 from MTα, and MT1, respectively . The CD spectra of AncTxs showed pronounced maxima and minima at 195 and 215 nm, respectively, typical of β-sheet structure. The CD spectrum of AncTx6 differed slightly from the other AncTxs with additional maxima and minima at 220 nm and 230 nm, that might reflect some local structural variations, as previously observed for MT123), MT2 (PDB id: 1FF4), MT7 (PDB id: 2VLW33) and ρ-Da1a (PDB id: 4IYE34) showed strong conservation of the backbone  was solved Fig. . As it mone Fig. . The RMSone Fig. . Loop 1 one Fig. .Figure 41A, α1B, α1D and α2C as compared to ρ-Da1a, MTβ and CM-3. Despite some minor modulations in the affinity constants of ancestral and actual toxins, the targeted receptors remained the same in this phylogenetic arm. However, for toxins located in the upper part of the phylogenetic tree , the receptor targeted and the selectivity profiles varied widely. Those toxin-receptor couples, that showed a significant radioligand displacement, were further analyzed by dose-response competition experiments in order to characterize precisely their selectivity profiles. The results are separated below along two evolutionary pathways to highlight changes in the affinity profile towards the various receptor subtypes that may have been the targets of the ancestral toxins.From the first screening with 1 and 5 µM of ancestral toxins final concentrations on various known targets of aminergic toxins, the first 4 ancestors (AncTx1 to AncTx4) display similar pharmacological properties on adrenoceptors α1A, α1B, α1D and α2C with affinity constants varying from nanomolar to micromolar range  and for the wild-type toxins ρ-Da1a (7.28), CM-3 (7.98) and MTβ (7.99) (Table AncTx1 showed the lowest affinity on the α9) Table , suggest9) Table .1B receptor, characterizes the pharmacological profiles of the different reconstructed toxins on the α2C receptor subtype. Indeed, AncTx1 interacted with the lowest affinity on the α2C receptor (pKi of 5.53) and a seven-fold gain in affinity occurred with the evolution to AncTx2 and AncTx3 with pKi values of 6.34 and 6.35, respectively. A minor improvement was achieved with AncTx4 that interacted with the α2C subtype with a slightly higher affinity (pKi = 6.83), surpassing its ancestors to reach values comparable to actual toxins: MTβ (pKi = 6.77), CM-3 (pKi = 6.78) and ρ-Da1a (pKi = 6.85)  Table .1D subtype, the pKi values for AncTx2 and AncTx3  were higher compared to AncTx1 (6.58), AncTx4 (6.39) also compared to modern toxins with affinity constants ranging widely from 5.95 for ρ-Da1a to 6.98 for CM-3  and those in ρ-Da1a (W28R + I38S) cause a substantial loss in affinity for α1D receptor (pKi dropping from 7.27 to 5.95) . Having recognized that the substitution (I38S) in AncTx3 affects α1D binding and that (W28R) increases α1B and α2C binding, the appropriate substitutions were integrated stepwise in AncTx1 to boost its selectivity. The selectivity profile for the variant AncTx1 I38S is characterized by pKi values of 9.33, 4.75, 6.44 and 4.90 for α1A, α1B, α1D and α2C subtypes, respectively . The pKi values for the AncTx1 double variant W28R-I38S were calculated to be 8.59, 6.41, 3.71 and 4.85 toward α1A, α1B, α1D and α2C respectively , the selectivity of this variant suffered from a large pKi gain toward α1B (6.41 vs. 4.75 for AncTx1 I38S). While ancestral toxin resurrection gave valuable binders with novel characteristics and pointed out key substitutions that can modulate aminergic toxins binding properties, combining this knowledge together in a rational manner to refine the pharmacological characteristics, ran into problems typical of classical rational and semi-rational protein engineering projects.A SAR for the α2B receptor, MT3 has the most divergent profile with an activity that spans seven of the nine receptors studied. MT3 interacts with the adrenoceptors α1A, α1B, α1D and α2C subtypes with comparable pKi values . MT3 recognized three extra targets compared to AncTx3, namely the α2A adrenoceptor and the muscarinic M1 and M4 subtypes with pKi values ranging from 6.22 for M1 to 9.08 for M4 . However, AncTx5 recognizes the dopamine D3 receptor, a subfamily of receptor targeted until now by only one aminergic toxin36. The pKi value of AncTx5 for this subtype was slightly higher (6.81) than for ρ-Da1b (6.12)  Table .Figure 8i values of 7.85, 6.66 and 7.44 for α2A, α2B and α2C respectively17. Nevertheless, AncTx5 with pKi values of 8.33, 8.11 and 8.46 on these same three subtypes was also, as for D3, a stronger binder than ρ-Da1b  and modifications at positions 26 (K to I) and 28 (W to R), already shown as key determinants for target modulation36.ρ-Da1b was first described for its interaction with the three α2 adrenoceptors with pK4, α1A, α1B, α1D, α2B and D3 may be derived from the changes in fingers II and III of these two proteins. As differences between AncTx3 and MT3 come from fingers I and II, we hypothesized that variations in affinity noted for M4, α2A, or α2C can be mapped to changes in these fingers.Due to the high number of substitutions at the other nodes  from MT1 to MTα are thought to be responsible for these functional differences. A single substitution marks the transition from AncTx6 to MTα. The parent shared the adrenoceptor α2B as its only target  was unexpected.AncTx6 represents the node that leads to MTα and MT1, two toxins that recently were shown to have a completely different pharmacological profile despite a 94% sequence identity37. This approach was recently applied using cDNA display on a three-finger scaffold, in order to produce novel molecules such as interleukin-6 receptor ligands21 and serine protease inhibitors22. Nevertheless, in order to obtain smaller but functionally rich libraries, rational protein engineering was developed by exploiting information on protein sequence, structure and function29. We applied this strategy on aminergic toxins using a loop permutation approach and create chimeric toxins with new functional profiles on muscarinic and adrenergic receptors23. Nevertheless, within a protein family with many functionally diverse members, a vertical strategy which reconstruct ancestral genes and study the effect of mutations on functional diversification should allow the identification of the structural determinants of protein function and the exploitation of this knowledge for protein engineering24.In the last decades, directed evolution was largely exploited as a protein engineering strategy to mimic natural protein evolution by an iterative process combining the generation of molecular diversity by random mutagenesis and the high throughput screening selection of library members with improved functions1A, α1B, α1D and α2C subtypes. Based on the AncTx4 pharmacological profile, the complementary role of positions 38 and 43 in the pharmacological selectivity of these toxins was reinforced to confirm previous conclusions made for ρ-Da1a, CM-3 and MTβ aminergic toxins36. Given that position 43 is located in the globular core of the toxin, a direct involvement in receptor interaction is unlikely. The reason for its impact is unclear but it may involve subtle structural effects. Interestingly, this region of 3FT has already been highlighted in a directed evolution work21. Despite a complete “randomization” of the tips of the three loops (23 amino acids out of 61), an additional mutation at position 44 occurred spontaneously in their most represented population of interleukin 6 receptor binders21. Regarding position 28, for the first time it was revealed crucial in adrenoceptors binding. This position is proximal to residue 38, confirming this region as key for adrenergic receptor binding. The mechanism behind the binding modulation at position 28 needs to be clarified but it might be due to a stabilizing effect at the tip of finger II as suggested for position 38 , we retained only six ancestral toxins (AncTx) with unique and intermediate primary structures relative to extant aminergic toxins. Interestingly, most of them also showed subtle and profound modulations in their pharmacological profiles. All these changes could then be directly correlated to amino acids substitutions. We also found three key positions in the ρ-Da1a evolutionary pathway  that can modulate by varying degrees the affinities for the α1B, α1D or α2C receptors with just two variants, unfortunately without achieving our primary goal to improve selectivity. Epistasis, a phenomenon already stated as important in protein evolution may have been the cause as revealed by other ancestral resurrection studies27. Indeed, the functional effects of a substitution could depend on its surrounding context or sequence background. Despite close homology, the same substitutions introduced in AncTx1 or AncTx3 did not result in either a decrease nor an increase in affinity for receptors α1A, α1B and α1D.On the basis of these freshly obtained SAR, new toxins variants with desired properties were engineered. We increased the affinity of AncTx1 for the α1A adrenoceptor subtypes. AncTx5 is the most potent aminergic toxin able to interact with the three α2 adrenoceptor subtypes. The recognition mechanism and binding mode of this toxin toward the three receptor subtypes become of great interest.In this study of ancestral toxins, we have obtained new, original and potentially useful functional profiles. AncTx1 is now the most selective peptide known for the αOur study contributes to the validation of the ancestral protein resurrection methodology that uses a small but functionally rich toxin library. Most of our results come from the first evolutionary pathway related to ρ-Da1a. Nonetheless, the second pathway opens a new promising line of investigation to understand the structure-activity relationships by delineating a few important positions.The aminergic toxins have proved to be good candidates for ancestral protein resurrection methodology as a small number of variants generated diverse pharmacological profiles. Undoubtedly, further studies will lead to methodology improvements derived from better evaluation of the sequence diversity and its link to the functional divergence before starting the resurrection project. The advantages over other protein engineering strategies are clear: a smaller number of variants with a more diverse activity profiles. Understanding the causes of the epistasis phenomenon would remove it as an obstacle and help future protein engineering work.et al.19, membrane preparations were obtained from transient (COS cells) or stable (CHO cells) cell lines prepared in the laboratory.All tritiated radioactive ligand were purchased from PerkinElmer Life Sciences . N-methylscopolamine, prazosin, yohimbine and spiperone were from Sigma-Aldrich . As previously described in Blanchet et al.36, the six AncTxs and the two engineered AncTx1 variants were synthesized on a 50 µmol scale using Fmoc chemistry on a Prelude synthesizer (Protein Technologies®) using HMPB ChemMatrix® resin functionalized with the appropriate protected amino acid (AA). Peptides were purified by reverse phase semi-preparative HPLC using a Vydac C18 column (250 × 10 mm) with an optimized gradient . The peptides were then subjected to folding in a buffer Tris-HCL 100 mM pH 8 containing 1 mM of EDTA, 1 mM of GSH and GSSG and 20% v/v of glycerol. The final reticulated peptides were purified using the same method described above and characterized by ESI MS (Bruker Esquire 3000 plus).As previously described in Blanchet et al.36, measurements were routinely performed at 20 °C in 0.1 cm path length quartz cells (Hellma) with a peptide concentration of 20.10−6 M in 5 mM sodium phosphate buffer pH 7.4. Spectra were recorded in the 190 to 260 nm wavelength range. Each graphic represents the average of three spectra.CD spectra were recorded on a Jasco J-815 CD spectropolarimeter. As previously described in Blanchet TM sitting drop vapor diffusion plates with 1 µl drops of protein and precipitant, stored in a constant temperature incubator at 20 °C. Limited screening for crystals was carried out starting from conditions used for MT733. Crystals of AncTx1-W28R/I38S were obtained from 1.9 M ammonium sulfate, 4% MPD, 2% 1,4-dioxane, 2% gamma-valerolactone, 0.132 M sodium citrate, pH 5.5. Four crystals were flash frozen in a cryoloop with a cryoprotectant consisting of 80% saturated lithium sulfate, 10% dioxane and 10% gamma-valerolactone. The crystals were shipped for automatic data collection at the Massif 1 at the ESRF in Grenoble, France on beamline ID30A-138. The XDS39 was used for data reduction with the script XDSME (https://github.com/legrandp/xdsme). The crystals belonging to the trigonal space-group P3121 with cell parameters a = b = 41.1 c = 67.1 Å diffracted to between 1.95 and 1.8 Å resolution. The structure was solved by molecular replacement starting from the ρ-Da1a crystal structure (PDB ID: 4IYE)34 with Phaser40. Refinement was carried out with REFMAC41 and Phenix42 and the electron density fitted with COOT43. Statistics for AncTx1-W28R/I38S structure are reported in Table Crystallization experiments were carried out with lyophilized toxin redissolved at 5 mg/ml in 1 M sodium acetate, pH 5.5. The crystallization trials were carried out using Cryst Chem1 to M5 muscarinic receptor subtypes, all three α1 and α2 adrenoceptor subtypes and the dopamine D3 subtype) by heterologous inhibition experiment as described in Blanchet et al.19. In short, 5 µM of each AncTxs were incubated (in duplicate) with a fixed amount of membrane preparation and a fixed concentration of a specific radioligand in 96-wells plate with 100 µl as final volume for 20h at RT. As previously described in Blanchet et al.19, binding experiments for muscarinic receptor subtypes, were carried out in saline phosphate buffer , pH 7.4, with 0.1% bovine serum albumin (BSA). [3H]-NMS was used as specific radioligand at 1 nM final concentration. The adrenergic α1 membranes were incubated in Tris buffer , pH 7.4, 0.1% BSA, with 1.6 nM of [3H]-Prazosin as radiotracer. For α2 adrenoceptors, binding was carried out in saline HEPES buffer , pH 7.4, 0.1% BSA and 1.7 nM of [3H]-Rauwolscine. Finally, binding assays on D3 dopamine receptor subtype were carried out in Tris buffer , pH 7.4, 0.1% BSA with 2 nM of [3H]-Methylspiperone. The affinity constants of the radioactive ligands for each membrane preparations were determined by equilibrium saturation experiments (n ≥ 2). Non-specific binding values were obtained by using N-methylscopolamine, prazosin, yohimbine and spiperone in excess (concentration up to 1000 times their Kd) in the binding assay on respectively: the muscarinic, α1, α2 and D3 receptor subtype membrane preparations. Binding reactions were stopped by filtration through GF/C filter pre-soaked in 0.5% polyethyleneimine on a cell harvester  and plates were dried. Ultimagold O  was added to each well and samples were counted using a Top-Count counter . After subtraction of the non-specific binding and normalization, only radiotracer displacement of 20% or more was considered as relevant and subjected to competition experiments.The six AncTxs were first screened on the 12 previously identified aminergic receptor targets reported for the aminergic toxins  were analyzed by non-linear regression analysis using Kaleidagraph 4.0 . After subtraction of the non-specific binding and normalization, a one-site inhibition curve was fitted to inhibition binding data, ICet al.19, sequences of aminergic three-finger toxins were retrieved from the UniProt Knowledgebase. Sequence alignments were carried out using MUSCLE from the MEGA5 package45. Alignments were visualized and inspected manually for eventual errors with the MEGA5 alignment visualizer and Jalview version 246 was used to edit the final colored alignment. The maximum likelihood method47 was used to reconstruct the phylogenetic trees. The best-fit substitution model, tree reconstruction, visualization, and annotation were chosen using MEGA5. Bootstraps support values were based on 10 000 replicates. Each ancestral amino acid and its associated probability value (P) at every site of each node were retrieved from an excel file supplied by MEGA5 and used to reconstruct the most probable ancestral sequence for each node of the tree. Few ambiguous sites were found, and to minimize the number of possible sequences, the amino-acid with the highest P was every time chosen.As described previously in Blanchet The position 38, located at the middle of the second loop, is quite closed to the tip of the loop II (known as a common “hot spot” for 3FTx to interact on their various targets) and may affect close and functionally important residues. Furthermore, as previously described, the S38I substitution could lead to a local stabilization of the Arg28, Arg40, Lys26 and Tyr36 lateral chains.Supplementary information


The similar UC efficiency of Yb:Er doped NaT(XO4)2 and β-NaYF4 compounds allowed testing equal subcutaneous depths of ex-vivo chicken tissue in both cases. This extraordinary behavior for NaT(XO4)2 oxides with large cutoff phonon energy (ħω≈ 920 cm-1) is ascribed to 4F9/2 electron population recycling to higher energy 4G11/2 level by a phonon assisted transition. Crystalline nanoparticles of Yb:Er:NaLu(MoO4)2 have been synthesized by sol-gel with sizes most commonly in the 50–80 nm range, showing a relatively small reduction of the UC efficiency with regards to bulk materials. Fluorescence lifetime and multiphoton imaging microscopies show that these nanoparticles can be efficiently distributed to all body organs of a perfused mouse.Yb and Er codoped NaT(XO An often used method for these purposes is the incorporation of inert nanoparticles (NPs) in thermodynamic equilibrium with the unknown system, acting as probes. Within this approach, NPs with up-conversion (UC) properties are recognized fluorescence nanoprobes because they are free of photobleaching shown by organic dyes  or photoimaging. ,13,143+ green UC). When combined with magnetic properties provided by the incorporation in the host of a paramagnetic ion, like Gd3+, optical and magnetic resonance multimodal imaging is possible.[A further outstanding characteristic of UCNPs is their intrinsic multifunctionality: An optical image can be obtained simultaneously to the measurement of a physical property  and transfers the absorbed energy to Er3+ by energy diffusion through the crystalline host. The origin of the singular UC efficiency of Yb:Er:β-NaYF4 has been attributed to the combination of good matching of Yb and Er energy level gaps, low host cutoff phonon energy (ħω≈ 500 cm-1), and presence of multisites for the Ln,[4 as the almost unique protagonist host.[Although UC is a phenomenon observed for different Ln  in virtually any Ln doped solid, Yb:Er:β-NaYFficiency. In this r the Ln, althoughist host.–234 as UC media, the search of alternative UC materials is continuously pursuit due to the following reasons: i) The synthesis of β-NaYF4 hexagonal gagarinite phase (4 cubic phase (S) of Yb:Er:β-NaYF4 is weak, in comparison to other studied materials; [4 is reduced by three orders of magnitude on going from bulk to ultrasmall NPs with ≈10 nm size.[Despite the prevalence of Yb:Er:β-NaYFterials;  and iv)  nm size.-1) can not achieve green Er3+ UC efficiencies comparable to that of fluorides because the 2H11/2+4S3/2 Er3+ electron population in oxides is strongly depleted by non radiative multiphonon relaxation to the low lying 4F9/2 Er3+ mutiplet, giving rise to strong red UC. This situation is also encountered for Yb:Er:Lu2O3 despite its lower cutoff phonon energy (ħω ≈ 650 cm-1).[Oxides have been studied as an alternative for UC purposes because of their superior chemical stability, easy synthesis and in most cases absence of phase transformations or polymorphism. However, after numerous studies it is generally believed that oxides with large cutoff phonon energy .S) in four members of the MT(XO4)2  tetragonal double molybdate (DMo) and double tungstate (DW) compound family: 10at%Yb:1at%Er:NaY(WO4)2, S(317 K) = 112×10−4 K-1;[4)2, S(317 K) = 120×10−4 K-1;[4)2, S(317 K) = 77×10−4 K-1;[4)2, S(317 K) = 160×10−4 K-1,[4, S(317 K) = 35×10−4 K-1.[The present work is motivated by recent reports of large UC ratiometric thermal sensitivity 2, S7 K = 1123+ multisites and/or structural disorder leading to a spatially variable distribution of crystal field potentials around Ln3+ are though to be key factors contributing to the large UC efficiency, and these structural characteristics are certainly shared by hexagonal β-NaYF4 and tetragonal (4)2 hosts, i.e. in the latter two different crystal sites, 2b and 2d, are randomly occupied by Na, T and Yb/Er cations. Neither earlier indicated works dealing with thermal sensitivity in polycrystalline MT(XO4)2 compounds nor UC characterizations of single crystals[Large UC ratiometric thermal sensitivity of used nanoprobes is an essential, but not sufficient, condition for any envisaged UCNP application, being large UC efficiency and suitable NP size simultaneous requisites. As previously indicated, the presence of Ln crystals,33 have 4)2 [4)2 [4)2,[4)2,[2 square sheets have been studied. These works show a significant spread of the reported S values which could be associated to the difference in material composition or to different experimental characterization techniques used in the involved laboratories.On the other hand, two of the previously cited works, NaY(WO4)2  and NaY(4)2 [4)2  deal wit4)2 [4)2, the morp4)2,[4)2, 50x50 nm4 with the same Yb and Er ion densities, as well as to determine the Yb and Er composition that optimizes the green UC efficiency in these hosts. We address this point by measuring the intensity of the output green photon flux at constant solid angle as well as by simulation of subcutaneous testing using ex-vivo breast chicken tissue and both types of compounds (DMo/DW and β-NaYF4). Moreover, although there are previous reports of sol-gel synthesis of MT(XO4)2 compounds, [4)2 NPs with diameter ranging 50–80 nm have been perfused in a mouse and their body distribution monitored with lifetime and multiphoton optical microscopies.Our purpose in the present work is to present a significant evaluation of the green UC efficiency for eight different DMo/DW hosts in direct comparison to the UC efficiency of Yb:Er:β-NaYFmpounds, ,34,35 in4. It is also shown that the green UC efficiency of Yb:Er doped Na-based DMo/DW is comparable to that found for Yb:Er:β-NaYF4, and that the UC efficiency reduction in the sol-gel synthesized UCNPs decreases only by a moderate factor of 10. This high UC efficiency allowed monitoring the sub-100 nm UCNPs in different body organs, including brain, of a perfused mouse. Finally, a thermal assisted energy transfer excitation mechanism is proposed as responsible for the extraordinary UC properties of DMo/DW compounds.We confirm that the thermometric properties of Yb:Er:DMo/DW compounds are superior to those of Yb:Er:β-NaYF4, have been made on the basis of micrometric sized powders prepared by solid state reaction and by hydrothermal (HT) synthesis. The sol-gel synthesis is exclusively used for the preparation of the DMo/DW UCNPs which are eventually perfused in a mouse.Comparisons of properties of different materials, either Yb:Er:DMo/DW or Yb:Er:β-NaYF2CO3 , WO3 , MoO3  and corresponding Y2O3 or Ln2O3  reagents were used for solid state reaction synthesis of DMo/DW compounds. The homogenized stoichiometric mixtures were firstly heated in air at 650°C (DMo) or 750°C (DW) for 18–24 h, cooled down to room temperature (RT), ground, and then heated at increasing temperatures up to 790°C (DMo) or 885°C (DW). The last thermal annealing to promote crystallization typically lasted 24–48 h.Na4)2 has been previously described in detail.[2MoO4·2H2O  or Na2WO4·2H2O  and the above indicated Y2O3 or Ln2O3 have been used as reagents. Briefly, Y or Ln nitrates were firstly prepared by dissolving together the required stoichiometric amounts of corresponding Y2O3 or Ln2O3 sesquioxides in a solution of nitric acid (10 ml of distilled water and 10 ml of 69% HNO3), which was heated until complete dryness. Then the Ln3+-nitrates were dissolved in 10 ml of distilled water, and a transparent solution of Na2(Mo/W)O4·2H2O with the stoichiometric amount of Mo/W in 10 ml of water was added. After 10 min of stirring the white suspension was transferred to a Teflon-lined autoclave, which was sealed and heated to 170°C for 24 h. The product resulting from the HT reaction was collected by centrifugation, washed several times with distilled water, and dried overnight at 125°C.The HT synthesis of Yb:Er:NaT(XOn detail. Na2MoO4·4 (7 mmol) samples  were synthesized by adding a previously prepared solution of Ln-nitrates to a clear ethanolic solution  of NaF , NaF:Ln = 4:1. The formed turbid suspension was stirred and its pH was adjusted to 3 by adding HF , and then treated at 220°C for 36 h in a Teflon-lined sealed autoclave. The obtained product was in each case collected by centrifugation and washed with ethanol several times, dried overnight at 125°C, and afterwards subjected to 2 h annealing at 300°C to remove defects associated to wet low-temperature synthesis methods, and to promote better crystallization.Yb:Er:β-NaYF2CO3, Y2O3, Ln2O3 reagents before described and either (NH4)6Mo7O24·4H2O (Fluka) or (NH4)6H2W12O40·nH2O (Alfa Aesar) were used as sources for the different required cations. The required amounts of Y2O3 or Ln2O3 raw materials were dissolved in 69% HNO3 and a suitable volume of deionized water under vigorous stirring and heating at 90°C. When the solution was transparent, Na2CO3, ammonium molybdate/tungstate, citric acid as complexing agent (CA) and ethyleneglycol (ET) were added to the solution. The molar ratio of chelated metal cations to the CA was 1:4 and the molar ratio of CA to ET was 1:10. The above mixture was stirred for 2 h at 80°C to get a stable precursor solution. Finally, some ammonia was added to adjust the pH value around 4–5. This solution was dried by slow heating at 120°C to obtain a black powder. NPs were obtained by calcination of this powder at 600–800°C temperature range. Higher (> 800°C) calcination temperatures lead to the formation of the Ln2O3 compounds, likely due to Na and Mo/W evaporation.[DMo/DW NPs were obtained by sol-gel following the modified Pechini method. Briefly, Naporation. In orderα radiation, and the Bragg diffracted light was collected by a Lynxeye detector. The crystalline domain size (d) was determined by the Scherrer formula, d = 0.9λ/Δ2θcosθ . For Yb:Er:NaLu(MoO4)2 we used the 112 Bragg reflection at 2θ≈ 28.5 deg.The phase and crystalline quality of the synthesized products was assessed by room temperature (RT) powder X-ray diffraction (pXRD). θ-2θ scans were performed on a Bruker AXS D8 Advance equipment under Cu KParticle morphology and its crystallinity were studied by transmission electron microscopy (TEM) and high resolution transmission electron microscopy (HRTEM) analyses carried out by using a 200 kV JEOL JEM 2100 and a 300 kV JEOL JEM3000F microscopes, respectively.The hydrodynamic size of the species present in the dispersions of sol-gel products were determined by dynamic light scattering (DLS) measurements with a Cordouan Technologies particle size analyzer, model Vasco 2.2. For UC emission evaluation the fluorescence was collected by a condenser lens (50 mm of diameter) parallel to the sample surface and focused to the entrance of a fiber ended Spectral Products compact spectrometer, model SM440. The residual excitation light was removed from the detection system by an Edmund Optics PO:7MOD-11081 short-wavelength pass filter with a transmission band between 402 nm and 838 nm. All spectra were corrected by the spectrometer background and its spectral response. The powdered samples were pressed and leveled to a reference horizontal plane and all UC measurements were collected under the same geometry to allow the evaluation of the relative efficiency. The UC efficiency was evaluated from the area under the green UC emissions. Standard optical absorption spectra were acquired with Varian, Cary 500, spectrophotometer.UC was excited at RT with the emission of a 25 A LIMO diode laser (DL). In order to minimize heating of the tested sample due to light absorption, the DL was operated in pulsed mode with a 10% duty cycle and the average current was limited to values lower than 7 A . Under these conditions the DL output is centered at λ = 973 nm (FWHM≈ 3 nm). The laser beam, delivered through an optical fiber, was focused by a f = 100 mm lens to a spot with ≈ 2 mm of diameter incident onto the sample at 45 deg. The average irradiation density was ≤ 4 W/cm2 and perfused transcardially first with a solution containing NPs followed by 4% paraformaldehyde in phosphate buffer saline  solution as described before.[Adult mice were deeply anesthetized with COd before. Several i microscope. Fluorescence was excited through a Nikon S Fluor 40× /0.9 NA DIC M/N2 dry objective. For excitation we used a Ti:Sapphire tunable Spectra Physics laser  coupled to an acousto-optic pulse picking modulator. NPs were excited at λ = 973 nm with a laser power of 1 nW at the sample and the emission was selected through a λ0 = 530±43 nm bandpass filter. Tissue images were acquired exciting at λ = 900 nm with a laser power of 450 nW at the sample, and the emission was selected through a λ0 = 445±20 nm bandpass filter. With a scan area of 256×256 pixels and a pixel dwell time of 32 μs, we could image a 160×160 μm2 area in sample at 625 nm pixel size and 0.625×0.625×1.000 μm3  voxel size.For FLIM we used an Alba laser-scanning module  interfaced with an inverted Nikon Eclipse TFLIM was performed following the digital frequency domain method and using a FLIM card previously described, which wa2, with a pixel size of 415 nm and a 0.415×0.415×1.000 μm3  voxel size. Three dimensional (3D) rendering was obtained from z-stacks of 38–45 optical sections with 2 μm step size under the same scanning conditions. IMARIS  and Fiji software were used for 3D rendering and post-processed composite images.Tiled 2-color multiphoton images were obtained on an upright LSM 780 Zeiss confocal microscope coupled to a Ti:Sapphire tunable Spectra Physics laser  and equipped with a deep in Plan Apochromat 20x /1 NA DIC 0,17 M27 75 mm water immersion objective. NPs were excited at λ = 973 nm with an average laser power of 8 nW at the sample, and the emission was collected through a spectral GaAsP photodetector in the λ = 517–562 nm range. Tissues were excited at λ = 850 nm with laser power of 600 nW at the sample, and the emission was collected by a photomultiplier tube in the λ = 600–650 nm range. Images of 2867×2867 pixels at a pixel dwell time of 1.52 μs were obtained by 3x3 tile scans to document sample areas of 1190×1190 μm2)4 and Yb:Er:β-NaYF4 compounds. 3+ electronic de-excitation from 2H11/2→4I15/2 (λ≈ 520–540 nm) and from 4S3/2→4I15/2 (λ≈ 540–560 nm) are observed. Additionally, other weaker UC emissions may be observed for the blue 2H9/2→4I15/2  and red 4F9/2→4I15/2 (λ≈ 630–700 nm) Er3+ transitions. It is worth noting that the relative intensity of the latter red UC emission is much lower in NaT(XO2)4 compounds than in β-NaYF4 one. This feature extends to all host compositions of the DMo/DW compounds studied and for any Yb and Er concentrations. Other notorious difference between the green UC spectrum in Yb:Er:DMo/DW and Yb:Er:β-NaYF4 is that the 2H11/2/4S3/2 UC intensity ratio is much larger in the formers.4 reference compound, similar Ln densities (in at/cm3) must be considered. For this purpose it must be taken into account that the tetragonal unit cell of DMo/DW, with a volume V≈ 300 Å3, contains two formula units (Z = 2),[4 fluoride , contains only one formula unit (Z = 1).[4)2 and β-NaYF4 hosts, respectively. The optimum dopant concentration for maximum Yb:Er:β-NaYF4 UC efficiency occurs for about 10at%Yb:1at%Er, while maximum UC efficiency in NaY(WO4)2 occurs at 25at%Yb:5at%Er substitution, i.e. shorter Ln-Ln distance is required in the scheelite host, likely associated to a lower host energy migration length.The efficiency of non radiative resonant transfer lies on donor-acceptor distance, thus in order to compare the UC efficiency of DMo/DW compounds with that of β-NaYF (Z = 2), while th (Z = 1). Thus sim4, taken as reference.In order to determine the most favorable DMo/DW host for Yb:Er green UC 4 and Yb:Er:DMo/DW compounds and as a proof of concept of its application for subcutaneous testing, we have monitored the UC attenuation in an ex-vivo chicken breast, As a further macroscopic evidence of the equivalence of UC efficiency in Yb:Er:β-NaYFI = I0et−κ), the attenuation coefficient results κ = 27 cm-1, independently of the DMo/DW or β-NaYF4 compound used as UC source. It is obvious that the determined κ value is much larger than the optical absorption coefficient of the breast chicken tissue at green and IR wavelengths, typically α = 0.4–0.8 cm-1 and α< 0.2 cm-1, respectively.[ex-vivo chicken breast tissue is composed of an increasing (with decreasing wavelength) background and some absorption peaks at specific wavelengths. The background is related to light scattering, therefore the UC attenuation observed in UC) is non-linearly related to the excitation pumping intensity, IP, i.e. IUC≈ IPn, n being typically equal to the number of photons involved in the UC mechanism (n = 1.5–3).ectively. In fact,2H11/2 and 4S3/2 Er3+ multiplets. Briefly, the change with temperature of the R = I(2H11/2)/I(4S3/2) UC intensity ratio determines the material UC ratiometric thermal sensitivity, S = dR/dT. More explicitly, R = C×exp(-ΔE/kBT) and S = R×(ΔE/kBT2), where kB is the Boltzmann constant and ΔE is the energy gap between 2H11/2 and 4S3/2 multiplets. C and ΔE are characteristics of each considered material and are usually calculated from LnR vs 1/T representations. 4)2 and 15at%Yb:1at%Er:β-NaYF4. The UC ratiometric thermal sensitivity obtained at T = 317 K (44°C) are S = 92×10−4 K-1 and S = 34×10−4 K-1, respectively. The high S value of the former is similar to those reported for other DMo/DW compounds.[r = σR/S (σR is the standard deviation of R) are 0.7 K and 1.8 K for the DW and β-NaYF4, respectively. Although thermal resolution is not a material property and precautions must be taken when comparing data obtained with different experimental setups, the present results show the advantage of using the DMo/DW instead of the reference fluoride for thermal measurements.Nanothermometry is a remarkable application of UCNPs, which is related to the Boltzmann electron population redistribution between the close in energy ompounds.–29 This -1K-1 at RT for a and c directions, respectively)[Nevertheless, it must be noted that thermometric measurements are complex due to several uncertainties. On the one hand, the considered DMo/DW materials have low thermal conductivity (1.47 and 1.62 Wmectively) and the 4)2 sample prepared by solid state reaction undergone three sequential heating/cooling round trip cycles, and S was evaluated independently for each one of the six up or down trips and for the whole data set, see S(317K) = 99×10−4 K-1 with an standard deviation  of σS(317 K) = 4.6×10−4 K-1 and the thermal resolution taking into account the whole data set was r = 0.9 K. Alternatively, when the resolution is calculated from ten measurements taken at the nominal constant temperature of 317 K its value is reduced to r = 0.65 K, what shows the importance of the calibration procedures for resolution calculation.In order to estimate a realistic uncertainty for the thermal parameters determined in this work we have conducted several additional experiments whose results are included in the SI. First, a 25at%Yb:5at%Er:NaY(WO4)2 sample prepared also by solid state reaction under extensive sequential heating at 850°C for 60 h and at 880°C for 3 h, see S decreases by less than 5% with increasing distance between the resistive heating element and the thermocouple union. However, it is worth noting that the sensitivity obtained for the latter sample with extensive thermal annealing is about 10% lower than that obtained for the previous preparation despite the same nominal sample composition. This shows the importance of material synthesis control for the DMo/DW considered which are prone to evaporate Na and Mo at high temperature (>800°C)[2(Mo/W)O4 and Ln2O3 phases in overheated polycrystalline materials.Moreover, measurements were made with the sensing thermocouple buried at different distances  from the heating element in the powder of a new 25at%Yb:5at%Er:NaY(WO (>800°C) leading 4)2 and 25at%Yb:5at%Er:NaLu(WO4)2, see DMo/DW NPs with optimum composition for green UC emission, i.e. 25at%Yb:5at%Er:NaLu(MoO4)2-intended products synthesized at short calcination times (< 6h) and low temperatures (≤ 750°C) show the presence of residual amounts of the Na2W2O7 phase (see 4)2 compounds, for which the scheelite phase is exclusively observed even after only 2 h of calcination at 600°C.The precursor black powders obtained by sol-gel were calcined to different temperatures  and times . The minimum calcination temperature (550°C) was determined by the need of full elimination of carbonaceous compounds see . Sol-gelhase see . This un4)2 sol-gel products after 4 h calcination at 600°C. At the lowest studied temperature (600°C) and for short calcination time (4 h) numerous quasi-spherical NPs with diameter in the 50–80 nm range appear detached or superimposed. For longer calcination times, 12 h, at the same temperature the NPs show a tendency to cluster but still their individual character is preserved. At higher calcination temperature, 700°C, some detached particles are observed even though their average size grows to 150–250 nm range, but often the particles are sintered forming rows. Grain boundaries between different particles are easily seen at this stage. Calcination at 800°C leads to general particle sintering forming superstructures with micrometric size. As indicated in 4)2 compounds preferred over NaT(WO4)2 counterparts due to the better phase purity upon 600°C calcination. A more general view of the effect of calcination temperature and time on the particle morphology is presented in the SI. As this point, it is worth to note that Yb:Er:NaLu(MoO4)2 NPs calcined at 600°C have a crystalline domain size of ≈40 nm, which is compatible with the HRTEM results described above.4)2 sol-gel products additionally processed with ultrasounds. To further confirm the water dispersibility of the NPs we have studied by DLS the size distribution of the species present in dispersions of the 600°C calcined 25at%Yb:5at%Er:NaLu(MoO4)2 NPs with average size 50–80 nm is reduced by a factor ≈10 with regards to micrometer sized products. This reduction is similar to that observed for 20at%Yb:2at%Er:β-NaYF4 NPs with size ≈ 100 nm.[4)2 NPs has been determined as S = 127×10−4 K-1, see 4.Based on the comparison of the UC output intensity, the green UC efficiency of sol-gel synthesized 25at%Yb:5at%Er:NaLu(MoO4)2 NPs obtained after calcination at 600°C (12 h) in an adult mouse. Afterwards, different mouse organs and tissues were examined. In order to explore the potential of the above NPs for body imaging and sensing we perfused the 25at%Yb:5at%Er:NaLu, indicating the presence of long-lived emissions, τ> 10 ns. With our experimental set-up  we are unable to precisely determine lifetime longer than 10 ns, thus the distribution corresponding to NPs is squeezed at the  polar coordinates. Nevertheless, the perfect superimposition of FLIM and intensity images clearly identifies the NPs entrapped in the examined lung section. The structure of the lung section analyzed in this example is shown in EXC = 900 nm and λEMI = 445±20 nm acquisition conditions. Under these conditions, the lifetime distribution close to  in the polar plot 2 compounds is controlled by the Yb3+ de-excitation rate with a lifetime in the 50 μs to 1 ms range, depending on Yb concentration and material microstructure,[Taking into account that the emission kinetic of Yb:Er:NaLu2  ħω = 917, 923, 919 cm-1, respectively.[4 reference compound.The green UC efficiency of Yb:Er:DMo/DW compounds is extraordinary large for what usually observed in oxides with large cutoff phonon energy. For NaT, however this process has low efficiency because of the small ground state absorption cross section of the 4I15/2 level (σABS≈ 0.3×10−20 cm2 for DMo/DW). Much more efficient excitation is obtained by Yb3+ (donor) photon absorption (in NaY(WO4)2 σABS = 1.84×10−20 and 2.52×10−20 cm2 for σ and π polarizations, respectively) and subsequent resonant energy transfer to Er3+ (acceptor), see 3+ and Er3+ have been reported in detail for several DMo/DW single crystals using low temperature (<10 K) spectroscopy.[4)2.[Ertroscopy.,53 Sinceopy.[4)2.3+ ground state absorption in DMo/DW occurs for the 0→0´ transition with maximum at 973 nm (10277.5 cm-1). It is interesting to note that the energy of the Er3+ 4I11/2 multiplet extends from 10200 to 10300 cm-1, thus a perfect Yb3+(2F5/2→2F7/2)-Er3+(4I15/2→4I11/2) resonance occurs. Moreover, the energy gap between Er3+ 4I11/2 and 4F7/2 multiplets in DMo/DW is 10276 cm-1, providing again a perfect resonance for the Yb3+(2F5/2→2F7/2)-Er3+(4I11/2→4F7/2) energy transfer. Standardly, in oxides the energy gaps between consecutive Er3+ multiplets below 4F7/2 are less than the total energy of two or three cutoff phonons, thus all these multiplets are populated by non radiative multiphonon relaxations[3+, 4F7/2→4I15/2), green , red , and infrared  UC emissions, notably with competitive intensities between green and red UC channels.[The most intense Yblaxations giving rchannels.,564S3/2 to 4F9/2, the almost complete depopulation of this red emitting level is achieved either by non radiative electronic relaxation or through electron recycling to upper excited states. Taking into account that the energy gap between 4F9/2 and 4G11/2 multiplets is in the 10895–11251 cm-1 range, it seems very likely that a phonon assisted resonant energy transfer Yb3+(2F5/2→2F7/2)+ħω(≈923 cm-1)-Er3+(4F9/2→4G11/2) and subsequent relaxation to 2H11/2 and 4S3/2 takes place, being this the origin of the large green UC efficiency found in Yb:Er:DMo/DW hosts. This electron recycling is illustrated in the inset of What is different in DMo/DW hosts is the extremely small intensity of red UC emission. This indicates that after non-radiative relaxation from 2H11/2+4S3/2 UC emission in Yb:Er:NaY(WO4)2 and Yb:Er:β-NaYF4, shown in 4, in the Yb:Er:NaY(WO4)2 case it increases from RT to 150°C and decays only for higher temperature. This shows that in the fluoride non radiative losses dominates the UC rate while in the considered oxide hosts the non radiative losses are diminished by host thermal energy transferred to excited electrons. Moreover, the n exponent of the IUC~IPn relationship, which is often identified with the number of photons involved in the UC excitation mechanism is larger for NaY(WO4)2 than for β-NaYF4, see SI.This proposal is further supported by the study of the thermal evolution of the total 4)2  compounds with crystalline disorder over the Na and T lattice sites exhibit green UC efficiency comparable to that of Yb:Er:β-NaYF4, generally accepted as reference compound for UC processes. This occurs in spite of the large cutoff phonon energy of these oxides (≈ 920 cm-1) which is about twice of that corresponding to the fluoride. It is suggested that the principal origin of such remarkable efficiency for an oxide host is the recycling of electrons in the 4F9/2 Er3+ multiplet by a phonon assisted transition to the 4G11/2 one. Such high UC yield in Yb:Er:DMo/DW allowed the subcutaneous testing of ex-vivo chicken breast with thickness similar (in excess of 2 mm) to that achieved with Yb:Er:β-NaYF4 material. Individual quasi-spherical nanoparticles with diameters in the 50–80 nm range and good crystalline quality have been obtained by the sol-gel method when the calcination temperature and time is limited to 600°C and 12 h, respectively, to prevent particle clustering and sintering observed at higher temperatures (700–800°C) and times (> 12 h). These experimental conditions are more easily fulfilled by NaT(MoO4)2 compounds having a high phase purity even with 600°C calcination, while NaT(WO4)2 counterparts require larger calcination temperature and time to fully eliminate the nucleation of Na2W2O7 phase. The UC signal of 25at%Yb:5at%Er:NaLu(MoO4)2 sol-gel NPs perfused in a mouse has been unequivocally identified by using fluorescence lifetime imaging microscopy (FLIM) and further compared with the SHG or autofluorescence images of the surrounding tissue. These studies show that the present sol-gel NPs with sub-100 nm size reach all mouse organs, including heart, lung, kidney, liver, spleen, eye and brain while keeping ≈10% of the UC efficiency observed in bulk counterpart material and offering unless 3 times larger UC ratiometric thermal sensitivity than Yb:Er: β-NaYF4 currently used UC nanoprobes.Yb:Er:NaT2  nanoparticles by the sol-gel method.Schematic chart of the preparation of NaLn(PDF)Click here for additional data file.S2 Fig0.5Yb0.5(WO4)2 nanoparticles. Heat flow (continuous line) and mass change (dashed line). The sample was heated and cooled in air at a rate of 10 K/min, with an isotherm period of 1 h at 720°C.DSC-TG analysis of the precursor powder prepared for the synthesis of NaLu(PDF)Click here for additional data file.S3 Fig4)2 sol-gel products obtained by calcination of the precursor black powder. (a) X = Mo. (b) X = W. X-ray reflections corresponding to the Na2W2O7 phase are labelled (*).Room temperature pXRD of 25at%Yb:5at%Er:NaLu(XO(PDF)Click here for additional data file.S4 Fig4)2 sol-gel synthesized nanoparticles after calcination at increasing temperatures and times. (a-c) Calcined at 600°C for 4h. Individual NPs are observed. (d-e) Calcined at 600°C for 12 h. A mixture of isolated and agglomerated NPs is observed. (f-h) Calcined at 700°C for 6h. Sintering of NPs is evident both in TEM and in HRTEM (right pictures). (i) Calcined at 800°C for 12h. Large scale sintering is observed.TEM and HRTEM images of 25at%Yb:5at%Er:NaLu(MoO(PDF)Click here for additional data file.S5 Fig4)2 sol-gel products synthesized after 6 h calcination at 600°C. The white solid powder was dispersed in distilled water and submitted to ultrasonic vibration for different times. a) As received dispersion. b) 4 min ultrasonic treatment. c) 8 min ultrasonic treatment. d) 12 min ultrasonic treatment.Effect of the ultrasonic treatment on the DLS-derived hydrodynamic size distributions of 25%Yb:5%Er:NaLu(MoO(PDF)Click here for additional data file.S6 Fig4)2 sol-gel products obtained after different calcination times at 600°C and subsequently treated 12 min in the ultrasonic processor. Products obtained after a) 6 h, b) 8h and c) 12 h of calcination.DLS-derived hydrodynamic size distributions of 25%Yb:5%Er:NaLu(MoO(PDF)Click here for additional data file.S7 Fig4)2 powdered sample prepared by solid state reaction. Three  consecutive heating (open symbols) /cooling (full symbols) cycles have been measured to show the thermal stability of the sample and to evaluate the reproducibility of the thermometric measurements. The line is the fit, LnR = 3.73–1277.85(1/T) (C = 41.7), of the whole data set providing S(317 K) = 99×10−4 K-1.LnR vs 1/T representation of the thermometric properties of a 25at%Yb:5at%Er:NaY(WO(PDF)Click here for additional data file.S8 Fig4)2 powdered sample prepared by solid state reaction. The lines are the fits for different data sets: h = 1.25 mm, black line, LnR = 3.614–1283.56(1/T), S(317 K) = 82.7×10−4 K-1; h = 1.9 mm, red line, LnR = 3.47–1227.92(1/T), S(317 K) = 81.6×10−4 K-1; and h = 2.4 mm, blue line, LnR = 3.39–1201.68(1/T), S(317 K) = 80.2×10−4 K-1.LnR vs 1/T representations obtained at three distances  between the heating plate and the tip of the thermocouple buried to determine the temperature of a 25at%Yb:5at%Er:NaY(WO(PDF)Click here for additional data file.S9 Fig4)2 sub-100 nm nanoparticles prepared by sol-gel . The particles were dispersed in distilled water for fluorescence measurements. The data fit provides LnR = 3.44–1020.83(1/T), C = 31.3, S(317K) = 127x10-4 K-1.LnR vs 1/T representation of the thermometric properties of 25at%Yb:5at%Er:NaLu(MoO(PDF)Click here for additional data file.S10 FigUC) as a function of the pump intensity (IP) for 15at%Yb:1at%Er:NaY(WO4)2  and 7.5at%Yb:0.5at%Er:NaYF4  compounds at (a) T = 20°C and (b) T = 150°C. The lines are the fits used to calculate the n exponent of the IUC~IPn relationship in the logarithmic representation.Evolution of the UC emission intensity (I(PDF)Click here for additional data file.S11 FigGeometric bidimensional representation of the lifetime distribution of a digital image.(PDF)Click here for additional data file.S12 FigEXC = 850 nm, λEMI = 600–650 nm. (b) UC image of NPs distributed in the same tissue area acquired λEXC = 973 nm, λEMI = 535 nm. (c) Composite image that allows the localization of the particles. Images are represented in pseudo-color normalized intensity scales: NPs in green and tissues in red.(a) Autofluorescence image of a lung section. λ(PDF)Click here for additional data file.S13 Fig4)2 NPs .3D rendering of z-scans showing the penetration of UC  inside a fragment of kidney, brain and liver tissues  after perfusion of the mouse with a PBS dispersion of sol-gel synthesized 25at%Yb:5at%Er:NaLu(MoO(PDF)Click here for additional data file.


The World Health Organization has developed ambient air quality guidelines at levels considered to be safe or of acceptable risk for human health. These guidelines are meant to support governments in defining national standards. It is unclear how they are followed.2.5, PM10, ozone, nitrogen dioxide, sulphur dioxide and carbon monoxide. We conducted literature and internet searches and asked country representatives about national ambient air quality standards.We compiled an inventory of ambient air quality standards for 194 countries worldwide for six air pollutants: PM2.5, PM10 and SO2 poorly complied with WHO guideline values. The agreement was higher for CO, SO2 (10-min averaging time) and NO2.We found information on 170 countries including 57 countries that did not set any air quality standards. Levels varied greatly by country and by pollutant. Ambient air quality standards for PMRegulatory differences mirror the differences in air quality and the related burden of disease around the globe. Governments worldwide should adopt science based air quality standards and clean air management plans to continuously improve air quality locally, nationally, and globally.The online version of this article (doi:10.1007/s00038-017-0952-y) contains supplementary material, which is available to authorized users. Ambient air pollution is hazardous to human health and caused an estimated 3.7 million premature deaths worldwide in 2012 WHO . A minor2.5 , PM10 , ozone (O3), nitrogen dioxide (NO2) and sulphur dioxide (SO2) , PM10 , O3 (1-h mean and 8-h average), NO2 (1- and 24-h average), SO2 , and CO . References and additional information (where necessary) are included in the table in the supplementary material  and on https://www.swisstph.ch/en/projects/ludok/grenzwerte/.For each country, we tabulated ambient air quality standards for different averaging times: PM2.5, PM10 and total suspended particles (TSP) were considered similar pollutants, as were NO2 and NOX. We considered that all averaging times between 1 and 24-h were similarly representative of regulations targeting short-term exposures to NO2, O3 and SO2, and that averaging times ≤1-h were similarly representative of ultra-short-term regulations for SO2. For all pollutants, standards set for averaging times >24-h  were considered representative for the regulation of long-term conditions. Wherever maximum allowable concentration (MAC) values or maximum permissible concentration (MPC) values were defined, we also listed these as “other” under the respective averaging time(s).We assumed that no standard was set for pollutants and/or averaging times that went unmentioned in the governmental documents. When more than one standard was defined for a single pollutant, we chose to list the standard that was most relevant for human exposure, e.g. standards for residential areas. The category “other” allowed us to indicate that a standard was set for a similar averaging time or pollutant, but a direct quantitative comparison was not possible. In this regard, PMWe quantified the number of countries that have set ambient air quality standards for at least one pollutant and averaging time for each region of the world, following the WHO’s geographical region classification WHO . We furtThe complete inventory table of national air quality standards for all pollutants and averaging times is shown in (Online Resource 1). We were able to identify whether air quality standards were set or not for 170 out of 194 countries  and NO2 (58–100%). Figure 10 and NO2. Maps for level of compliance for annual mean concentrations of PM2.5 and SO2 can be found in the Online Resource 3 and 4. Maps for short-term averaging times can be found in Online Resource 5 (PM10), 6 (PM2.5), 7 (SO2), 8 (NO2), 9 (O3), 10 and 11 (CO).The pollutants most often regulated with short-term standards (averaging time ≤24-h) are ambient daily averages of PM3 for 24-h PM10 was met by 47 countries including Malawi with an even more stringent standard of 25 µg/m3. The six countries Burkina Faso, Botswana, Benin, Senegal, Uzbekistan and Saudi Arabia have set higher standards than the WHO Interim-Target 1 which was set at 150 µg/m3. Canada was the only country that did not regulate PM10 but PM2.5, only. Seven countries out of 100 adopted the daily mean WHO guideline value of 20 µg/m3 for SO2. Regarding NO2 standards for 1-h daily maximum 56 out of the 77 countries that have set standards complied at least with the standard proposed by the WHO.The guideline value of 50 µg/m2 was the most often regulated pollutant with 85 countries of which 56 complied with the WHO guideline value set at 40 µg/m3 as the annual mean concentration. Lower limits (30 µg/m3) were set by Switzerland, Austria and Mongolia. Long-term standards for PM10 and/or PM2.5 were set by 83 countries. Seven countries regulated PM2.5 only without standards for PM10 . The PM10 WHO annual guideline value of 20 µg/m3 was adopted by six authorities , whereas most countries have set values between 40 and 50 µg/m3 up to 120 µg/m3. Annual mean PM2.5 limits were set by 62 countries at around 25 µg/m3 as opposed to the WHO guideline value of 10 µg/m3, met by six countries only, namely Australia and Malawi with a 20% lower standard (8 µg/m3) and Afghanistan, Cameroon, Canada and Iran.NO3, NO2 and CO. We also provide an update on short- and long-term standards set for PM2.5, PM10 and SO2. We were able to compile information about ambient air quality standards from 170 countries from all continents. The standards varied greatly between countries, despite universally generalizable scientific evidence of the substantial and serious health effects of ambient air pollution. The discrepancy in dealing with scientific evidence reflects the diversity in abilities and priorities of policy makers to regulate air quality and to implement policies that aim at reducing air pollution and protecting health. According to the WHO, its guideline values aim at air quality with only little or no effects for human health. As acknowledged by the WHO, our study confirms that it is not only public health concerns and scientific evidence driving the setting of air quality standards but also perceptions about technological feasibility, economic constraints, political pressure and social factors