Resultados de investigación

Multimedia component 1., Peer reviewed

Peer reviewed

Synthesis of intermediates 5, 6, 8a,b, 9a,b, 10b,c, and 11b S4 In vitro and in vivo biological methods S8 Figure S1. Best scoring poses for 12c in each of the AChE models evaluated S17 Figure S2. Interactions of the 6-chlorotacrine moiety of 12c in the CAS of AChE S17 Figure S3. RMSD values of the 6-chlorotacrine moiety of 12c over the last 100 ns of each simulation S18 Figure S4. RMSD values of the TPPU moiety of 12c over the last 100 ns of each simulation S18 Figure S5. Binding mode of donepezil in control simulations S19 Figure S6. In vivo treatment and experimental timeline S19 Figure S7. Familiarization phase of the NORT test S19 Table S1. Summary of the AChE structures available in the PDB that were analyzed to identify prior knowledge of the cryptic pocket in the PAS S20 Table S2. Reported and experimental PAMPA-BBB permeability of the commercial drugs used for assay validation S21 Table S3. Quantities of reagents, microsomes, and test compounds used in the microsomal stability assays S22 Table S4. Gradient used in the UPLC-MS/MS analysis for microsomal stability studies S22 Table S5. Antibodies used in Western Blot (WB) S23 Table S6. Primers and probes used in qPCR studies S23 Table S7. Reference c1 and c2 values for Trp286 rotamers in AChE S24 References S25 Copies of 1H and 13C NMR spectra and HPLC traces of target compounds S28, Peer reviewed

Procedure for tapering Au wires; additional HRTEM image; details of the calculation for the possible dimensions of a coherent domain enabled by large radii of curvature of concentric graphenes; tentative growth scenario for the cone parts; further data on modeling., Peer reviewed

Normalized calculation for SAR values and magnetization relative to the (Fe + Mn) content of the ferrite; FT-IR spectra for S2 and NF NPs, high-resolution XPS spectra and table with the peak position of the Mn 3s orbital for S1, S2, and NF NPs, table with the Mössbauer parameters derived from the spectra recorded at 5 and 270 K, respectively, for the samples under study, and table with AC data fitted to the Arrhenius model; and heating curves of temperature increase as a function of time for the four samples at different frequencies (100 kHz up to 300 kHz) and 16 kA/m., Peer reviewed

Supplementary Table 1: Primers used in this work., Steryl esters (SE) are stored in cytoplasmic lipid droplets and serve as a reservoir of sterols that helps to maintain free sterols (FS) homeostasis in cell membranes throughout plant growth and development, and provides the FS needed to meet the high demand of these key plasma membrane components during rapid plant organ growth and expansion. SE are also involved in the recycling of sterols and fatty acids released from membranes during plant tissues senescence. SE are synthesized by sterol acyltransferases, which catalyze the transfer of long-chain fatty acid groups to the hydroxyl group at C3 position of FS. Depending on the donor substrate, these enzymes are called acyl-CoA:sterol acyltransferases (ASAT), when the substrate is a long-chain acyl-CoA, and phospholipid:sterol acyltransferases (PSAT), which use a phospholipid as a donor substrate. We have recently identified and preliminary characterized the tomato (Solanum lycopersicum cv. Micro-Tom) SlASAT1 and SlPSAT1 enzymes. To gain further insight into the biological role of these enzymes and SE biosynthesis in tomato, we generated and characterized CRISPR/Cas9 single knock-out mutants lacking SlPSAT1 (slpsat1) and SlASAT1 (slasat1), as well as the double mutant slpsat1 x slasat1. Analysis of FS and SE profiles in seeds and leaves of the single and double mutants revealed a strong depletion of SE in slpsat1, that was even more pronounced in the slpsat1 x slasat1 mutant, while an increase of SE levels was observed in slasat1. Moreover, SlPSAT1 and SlASAT1 inactivation affected in different ways several important cellular and physiological processes, like leaf lipid bo1dies formation, seed germination speed, leaf senescence, and the plant size. Altogether, our results indicate that SlPSAT1 has a predominant role in tomato SE biosynthesis while SlASAT1 would mainly regulate the flux of the sterol pathway. It is also worth to mention that some of the metabolic and physiological responses in the tomato mutants lacking functional SlPSAT1 or SlASAT1 are different from those previously reported in Arabidopsis, being remarkable the synergistic effect of SlASAT1 inactivation in the absence of a functional SlPSAT1 on the early germination and premature senescence phenotypes., Peer reviewed

Resources available on the publisher's site: http://dx.doi.org/10.1182/blood.2021012805, Peer reviewed

Experimental section including preparation of MoSe2, f-MoSe2, PorMn, and MoSe2–MnP, high-magnification HAADF-STEM images, UV–vis spectra, and electrochemical data., Peer reviewed

Table S2. FS and SE quantification in seeds of wt, slasat1, slpsat1 and slpsat1 x slasat1 mutants. Data are shown as average values from three biological replicates with SEM in parentheses. n.d. stands for not detected. Significant changes compared to wild-type (WT) seeds are indicated by asterisks (*P<0.05; **P<0.01; ***P<0.005)., Steryl esters (SE) are stored in cytoplasmic lipid droplets and serve as a reservoir of sterols that helps to maintain free sterols (FS) homeostasis in cell membranes throughout plant growth and development, and provides the FS needed to meet the high demand of these key plasma membrane components during rapid plant organ growth and expansion. SE are also involved in the recycling of sterols and fatty acids released from membranes during plant tissues senescence. SE are synthesized by sterol acyltransferases, which catalyze the transfer of long-chain fatty acid groups to the hydroxyl group at C3 position of FS. Depending on the donor substrate, these enzymes are called acyl-CoA:sterol acyltransferases (ASAT), when the substrate is a long-chain acyl-CoA, and phospholipid:sterol acyltransferases (PSAT), which use a phospholipid as a donor substrate. We have recently identified and preliminary characterized the tomato (Solanum lycopersicum cv. Micro-Tom) SlASAT1 and SlPSAT1 enzymes. To gain further insight into the biological role of these enzymes and SE biosynthesis in tomato, we generated and characterized CRISPR/Cas9 single knock-out mutants lacking SlPSAT1 (slpsat1) and SlASAT1 (slasat1), as well as the double mutant slpsat1 x slasat1. Analysis of FS and SE profiles in seeds and leaves of the single and double mutants revealed a strong depletion of SE in slpsat1, that was even more pronounced in the slpsat1 x slasat1 mutant, while an increase of SE levels was observed in slasat1. Moreover, SlPSAT1 and SlASAT1 inactivation affected in different ways several important cellular and physiological processes, like leaf lipid bo1dies formation, seed germination speed, leaf senescence, and the plant size. Altogether, our results indicate that SlPSAT1 has a predominant role in tomato SE biosynthesis while SlASAT1 would mainly regulate the flux of the sterol pathway. It is also worth to mention that some of the metabolic and physiological responses in the tomato mutants lacking functional SlPSAT1 or SlASAT1 are different from those previously reported in Arabidopsis, being remarkable the synergistic effect of SlASAT1 inactivation in the absence of a functional SlPSAT1 on the early germination and premature senescence phenotypes., Peer reviewed

Table S3. FS and SE quantification in leaves of wt, slasat1, slpsat1 and slpsat1 x slasat1 mutants. Data are shown as average values from four biological replicates with SEM in parentheses. n.d. stands for not detected. Significant changes compared to wild-type leaves are indicated by asterisks (*P<0.05; **P<0.01; ***P<0.005)., Steryl esters (SE) are stored in cytoplasmic lipid droplets and serve as a reservoir of sterols that helps to maintain free sterols (FS) homeostasis in cell membranes throughout plant growth and development, and provides the FS needed to meet the high demand of these key plasma membrane components during rapid plant organ growth and expansion. SE are also involved in the recycling of sterols and fatty acids released from membranes during plant tissues senescence. SE are synthesized by sterol acyltransferases, which catalyze the transfer of long-chain fatty acid groups to the hydroxyl group at C3 position of FS. Depending on the donor substrate, these enzymes are called acyl-CoA:sterol acyltransferases (ASAT), when the substrate is a long-chain acyl-CoA, and phospholipid:sterol acyltransferases (PSAT), which use a phospholipid as a donor substrate. We have recently identified and preliminary characterized the tomato (Solanum lycopersicum cv. Micro-Tom) SlASAT1 and SlPSAT1 enzymes. To gain further insight into the biological role of these enzymes and SE biosynthesis in tomato, we generated and characterized CRISPR/Cas9 single knock-out mutants lacking SlPSAT1 (slpsat1) and SlASAT1 (slasat1), as well as the double mutant slpsat1 x slasat1. Analysis of FS and SE profiles in seeds and leaves of the single and double mutants revealed a strong depletion of SE in slpsat1, that was even more pronounced in the slpsat1 x slasat1 mutant, while an increase of SE levels was observed in slasat1. Moreover, SlPSAT1 and SlASAT1 inactivation affected in different ways several important cellular and physiological processes, like leaf lipid bo1dies formation, seed germination speed, leaf senescence, and the plant size. Altogether, our results indicate that SlPSAT1 has a predominant role in tomato SE biosynthesis while SlASAT1 would mainly regulate the flux of the sterol pathway. It is also worth to mention that some of the metabolic and physiological responses in the tomato mutants lacking functional SlPSAT1 or SlASAT1 are different from those previously reported in Arabidopsis, being remarkable the synergistic effect of SlASAT1 inactivation in the absence of a functional SlPSAT1 on the early germination and premature senescence phenotypes., Peer reviewed