Drosophila. Ultrastructural analysis of σ2-adaptin mutants show an accumulation of large vesicles and membranous structures akin to endosomes at the synapse. We found that mutations in σ2-adaptin lead to an accumulation of Tkv receptors at the presynaptic membrane. Interestingly, the level of small GTPase Rab11 was significantly reduced in the σ2-adaptin mutant synapses. However, expression of Rab11 does not restore the synaptic defects of σ2-adaptin mutations. We propose a model in which AP2 regulates Tkv internalization and endosomal recycling to control synaptic growth.Compromised endocytosis in neurons leads to synapse overgrowth and altered organization of synaptic proteins. However, the molecular players and the signaling pathways which regulate the process remain poorly understood. Here, we show that σ2-adaptin, one of the subunits of the AP2-complex, genetically interacts with Mad, Medea and Dad  to control neuromuscular junction (NMJ) growth in  Our study contributes to a broader understanding of AP2 complex-dependent regulation of synaptic growth signaling, which might have implications in neurodevelopment under conditions where endocytosis and trafficking of the BMP receptors are perturbed.Understanding the regulation of synaptic development and refinement could provide mechanistic insights into the neural basis of fundamental processes such as learning, memory and adaptation. In this study, we describe the role of σDrosophila neuromuscular junction (NMJ) synapses is a well-studied growth-promoting pathway (Drosophila) and its translocation into the motor neuron nuclei, followed by the transcription of BMP target genes , Wingless (Wnt), and Ubiquitin-Proteasome-mediated protein degradation  pathways . Besides pathway . The canet genes .Drosophila NMJ, the retrograde BMP signaling is initiated by secretion of Glass bottom boat (Gbb) from the muscle and the motor neurons  and aberrant synaptic growth. For instance, it is not known whether specific modes of endocytosis externalize specific cargos whose trafficking defect perturbs synaptic signaling. CME, the major endocytic pathway, is required not only for basal synaptic transmission at nerve terminals but also for synapse development . For insosophila . It is uosophila .In central synapses, AP2-dependent CME is dispensable for membrane regeneration from the presynaptic plasma membrane following high-frequency nerve stimulation . Howeverσ2-adaptin mutants : 7tkv (BL3242), 13Med (BL7340), A12wit (BL5173), j1E4Dad (BL10305), KG02457AP2σ (BL13478), UAS-Rab11-GFP (BL8506), k00237Mad (BL10474), α-adaptin dsRNA (BL32866), Clc dsRNA (BL27496), 1Synj (BL24883), 2Synj (BL24884). Other lines used in this study are: D42-Gal4  to expose the NMJs and fixed in 4% paraformaldehyde in PBS (pH 7.2) for 30 min at room temperature. Fillets were then washed in PBS containing 0.15% Triton X-100, blocked for 1 h with 5% bovine serum albumin (BSA), followed by overnight incubation with primary antibody at 4°C. The monoclonal antibody anti-CSP (1:100) was obtained from the Developmental Studies Hybridoma Bank (DSHB). The polyclonal antibody against Rab5  were recorded for 60 s, followed by recordings of EJPs at 1 Hz stimulation. For High-frequency recording, nerves were stimulated at 10 Hz, and EJPs were recorded for 5 min. For recording EJPs, stimulation pulse was delivered using Grass S88 stimulator . The signals were amplified using Axoclamp 900A, digitized using Digidata 1440A, and acquired using pClamp10 software (Molecular Devices). Muscles with resting membrane potential between −60 and −75 mV were used for analysis. The data were analyzed using the Mini Analysis program .All intracellular recordings were performed on wandering third instar larvae as described previously . BrieflyConfocal images of muscle four NMJ at A2 hemisegment were used to plot the intensity profile. A single bouton section was used, and the intensities of Tkv and HRP were analyzed by drawing a line across the bouton using Fiji/ImageJ software. The graph was plotted in Microsoft Excel using the intensity values obtained from Fiji/ImageJ software. As the intensity of Tkv in control was too low to plot the graph, all the intensity values were multiplied two-fold.m cacodylate buffer containing 2% glutaraldehyde for 10 min at room temperature, transferred to a fresh fixative, and kept overnight at 4°C. The fillets were postfixed for 1 h with 2% osmium tetroxide (OsO4) solution prepared in 0.12 m cacodylate buffer. The samples were rinsed with 0.12 m cacodylate buffer followed by washes with distilled water to avoid precipitation of cacodylate with Uranyl acetate. Subsequently, the samples were subjected to en bloc staining with 2% uranyl acetate. The stained fillets were again washed with distilled water and dehydrated using graded solutions of ethanol before final infiltration of the samples through propylene oxide for 30 min. Stained and dehydrated fillets were embedded in epoxy resin and hardened overnight at 60°C. Muscles embedded in epoxy resin were sectioned at 60 nm. Ultrathin sections of the muscles stained with 2% uranyl acetate (in 70% ethanol) and 1% aqueous lead citrate were examined at 120 KV on a Tecnai G2 Spirit BioTWIN (FEI) electron microscope. The number of synaptic vesicles per bouton were counted manually using the Multi-point tool in ImageJ/Fiji software and then divided by their respective bouton areas to obtain the vesicle density/μm2 area of a bouton. For vesicle size, diameters of at least 100 vesicles from 10 bouton sections of each genotype were used for quantification. For calculating the number of synaptic vesicles docked at the active zones, only those vesicles that were touching the T-bar platform were counted. For calculating the SSR thickness, the scale bar in the images was first calibrated to the number of pixels using the Set scale function in ImageJ/Fiji software. This was followed by using the Straight-line tool to draw a line across the SSR and Measure tool to calculate the thickness.TEM was performed as described previously . Brieflym Tris-HCl, pH 6.8, 25 mm KCl, 2 mm EDTA, 0.3 m sucrose, and 2% SDS in water. The homogenized sample was then mixed with an equal volume of 2× Laemmli buffer. The protein equivalent to 50 μg was separated on 12% SDS-PAGE and transferred to Hybond-LFP PVDF membrane . The membrane was then blocked with 5% skimmed milk for 1 h, followed by overnight incubation with anti-Rab11 (1:2000) and anti-Tubulin (1:5000) antibody. IRDye 800 (1:10 000) was used as a secondary antibody, and signals were visualized on LI-COR Odyssey platform. The density of Western bands was quantified using ImageJ/Fiji software.The western blot analysis was done as previously described previouusly . Brieflypost hoc Tukey’s test, or Student’s t test was used. GraphPad Prism 8 was used to plot the graph. Error bars in all the histograms represent SEM. *p < 0.5, **p < 0.01, ***p < 0.001.For fluorescence quantification, images were captured using a laser scanning confocal microscope . All the control and experimental fillets were processed in the same way, and the fluorescence images were captured under the same settings for every experimental set. For bouton quantification, CSP-labeled structures were counted at muscle 6/7 of A2 hemisegment. The number of boutons from each NMJ was normalized to the respective muscle area. To calculate the bouton number, NMJs from A2 hemisegment were captured using a plan apochromat 40× objective, 1.4 NA and all the CSP positive boutons were counted manually in ImageJ/Fiji software. For muscle area quantification, images from A2 hemisegment were captured using 20× objective, and the area was quantified using ZEN2 software . For bouton number quantification, the total number of boutons per NMJ was divided by the respective muscle areas. For fluorescence intensity quantification, NMJs from muscle four were captured using a plan apochromat 60×, 1.4 NA objective. For each NMJ, the fluorescence intensity from each bouton was subtracted from the background intensity, and the average intensity was normalized to the control. The fluorescence intensity was calculated using ImageJ/Fiji software. For bouton area quantification, NMJs from muscle 6/7 at A2 hemisegment were captured, and the area was calculated by drawing a free-hand sketch around CSP positive bouton using ImageJ/Fiji software. For multiple comparisons, one-way ANOVA followed by σ2-adaptin mutants caused NMJ overgrowth and upregulation of pMad, an effector of the BMP pathway. Two previously characterized σ2-adaptin alleles, ang7,AP2σ a strong hypomorph and KG02457AP2σ, a null allele was used in the current study , 13, AP2σKG02457/AP2σang7Med , and k00237Mad/+; KG02457/AP2σang7AP2σ  when compared with KG02457AP2σ/ang7AP2σ . However, there was no significant difference between 1118w control, heterozygous KG02457AP2σ/+, and 7tkv/+ , KG02457/AP2σang7AP2σ (B), A12wit, KG02457/AP2σang7AP2σ (C), double immunolabeled with a presynaptic vesicle marker, CSP (magenta), and a neuronal membrane marker, HRP (green), to mark the bouton outline. Reducing the levels of BMP Type II receptor in the KG02457/AP2σang7AP2σ background reduces the synaptic overgrowth. Scale bar in C represents 10 μm. D, Histogram showing the average bouton number normalized to the muscle area from muscle 6/7 NMJ at A2 hemisegment in control animals (1.33 ± 0.03), KG02457/AP2σang7AP2σ (2.50 ± 0.11), and A12wit, KG02457/AP2σang7AP2σ (2.0 ± 0.07). Error bar represents SEM; the statistical analysis was done using one-way ANOVA followed by post hoc Tukey’s test. **p < 0.01, ***p < 0.001. Download Figure 1-1, TIF file.Reducing levels of Wit receptor rescues the bouton number in wit significantly reduced the σ2-adaptin induced synaptic overgrowth , 13Med , and k00237Mad/+; KG02457/AP2σang7AP2σ  in σ2-adaptin mutant background partially restored the bouton area when compared with σ2-adaptin mutant alone ] and Dad [j1E4/+Dad (1.39 ± 0.09)] was comparable with 1118w control (1.26 ± 0.06), transheterozygous j1E4/AP2σKG02457Dad  showed significantly higher bouton number when compared with 1118wcontrols (σ2-adaptin and Dad mutants j1E4/AP2σKG02457Dad (8.51 ± 0.61) when compared with 1118wcontrol  significantly rescued the synaptic overgrowth in σ2-adaptin mutant ang7/AP2σKG02457AP2σ, quantal content (QC), and high-frequency intracellular recording on 1118w control, heterozygous 7/+tkv, KG02457/AP2σang7AP2σ, and 7/+; AP2σKG02457/AP2σang7tkv larvae  were not significantly different from σ2-adaptin mutant KG02457/AP2σang7AP2σ stimulations  and increased size of the SVs in the σ2-adaptin mutants . While there was no change in the number of SVs docked at the active zones , we found significantly decreased thickness of the subsynaptic reticulum  in the σ2-adaptin mutants. These ultrastructural defects were rescued on ubiquitous expression of a σ2-adaptin transgene  or clathrin light chain  in motor neurons showed significantly increased synaptic Tkv levels  or the late endosomal marker, Rab7 (1118w: 100.0 ± 5.71 vs KG02457/AP2σang7AP2σ: 115.6 ± 8.17) were not altered in σ2-adaptin mutant synapses  in σ2-adaptin mutant synapses , KG02457/AP2σang7AP2σ (102.8 ± 11.07), D42-Gal4, ang7AP2σ/KG02457UAS- AP2σ, AP2σ (84.45 ± 6.11), and ex2/93BiRab11 (65.06 ± 7.16). Error bar represents SEM; the statistical analysis was done using one-way ANOVA followed by post hoc Tukey’s test. *p < 0.05; ns, not significant. Download Figure 6-1, TIF file.Drosophila NMJ  compared with control animals . However, the Tkv enrichment pattern was not the same as that in σ2-adaptin mutants  and presynaptic membrane marker HRP (magenta) in D42-Gal4/UAS-tkv-EGFP  and D42-Gal4/UAS-tkv-EGFP . The intensity profile across the bouton in D42-Gal4/UAS-tkv-EGFP (F) suggests that Tkv is not enriched at the presynaptic membrane. Download Figure 7-1, TIF file.Tkv-EGFP is not localized at the presynaptic membrane in the σ2-adaptin mutants showed reduced Rab11 levels at the NMJ, we next asked whether Rab11 was responsible for the morphologic defects in these mutants. To assess this, we first examined whether reducing Rab11 levels phenocopied σ2-adaptin mutations. Interestingly, we found that Rab11 mutants or neuronal expression of a dominant-negative form of Rab11 (S25NRab11) indeed phenocopied the NMJ morphologic defects in σ2-adaptin mutants and showed NMJ overgrowth  did not prevent the synaptic overgrowth  failed to prevent the synaptic overgrowth in σ2-adaptin mutants , D42-Gal4-driven UAS-Rab11-GFP (B), and D42-Gal4-driven Q70L-GFPUAS-Rab11 (C) double immunolabeled with a presynaptic vesicle marker, CSP (magenta), and a neuronal membrane marker, HRP (green), to mark the bouton outline. Expressing the wild-type and active form of Rab11 does not show morphological changes at the synapse. Scale bar in C represents 20 μm. D, Histogram showing the average bouton number normalized to the muscle area from muscle 6/7 NMJ at A2 hemisegment in control animals (1.56 ± 0.06), UAS-Rab11-GFP/+; D42-Gal4/+ (1.56 ± 0.07), and Q70L-GFPUAS-Rab11; D42-Gal4/+ (1.58 ± 0.09). Error bar represents SEM; the statistical analysis was done using one-way ANOVA followed by post hoc Tukey’s test. ns, not significant. Download Figure 8-1, TIF file.Expressing a wild-type or constitutively active form of Rab11 does not alter the synaptic morphology. 10.1523/ENEURO.0044-22.2022.f8-2Extended Data Figure 8-2σ2-adaptin mutants. A–D, Confocal images of NMJ synapses at muscle 6/7 NMJ at A2 hemisegment showing synaptic growth in (A) Control animals, (B) CAD42-Gal4 > Rab11, (C) KG02457/AP2σang7AP2σ, and (D) CA, AP2σang7/AP2σKG02457D42-Gal4 > Rab11 double immunolabeled with a presynaptic synaptic vesicle marker, CSP (magenta) and a neuronal membrane marker, HRP (green) to mark the bouton outline. Scale bar in D represents 20 μm. E, Histogram showing average bouton number normalized to the muscle area from muscle 6/7 NMJ at A2 hemisegment in control animals (1.05 ± 0.11), CAD42-Gal4 > Rab11 (1.4 ± 0.10), KG02457/AP2σang7AP2σ (2.9 ± 0.14), and CA, AP2σang7/AP2σKG02457D42-Gal4 > Rab11 (2.5 ± 0.14). Error bar represents SEM; statistical analysis was done using one-way ANOVA followed by post hoc Tukey’s test. **p < 0.01; ns, not significant. Download Figure 8-2, TIF file.Constitutively active Rab11 does not restore the NMJ morphological defects of 10.1523/ENEURO.0044-22.2022.f8-3Extended Data Figure 8-3σ2-adaptin. A, Representative traces of mEJP in control, DND42 > Rab11, WTD42 > Rab11, heteroallelic KG02457/AP2σang7AP2σ, DND42 > Rab11, KG02457/AP2σang7AP2σ, and WTD42 > Rab11, KG02457/AP2σang7AP2σ larvae. B, Representative tracesof EJP in control, DND42 > Rab11, WTD42 > Rab11, heteroallelic KG02457/AP2σang7AP2σ, DND42 > Rab11, KG02457/AP2σang7AP2σ, and WTD42 > Rab11, KG02457/AP2σang7AP2σ larvae. C, Quantification of average EJP amplitude in control (44.42 ± 1.93), DND42 > Rab11 (53.89 ± 2.35), WTD42 > Rab11 (52.71 ± 2.42), heteroallelic AP2σKG02457/AP2σang7 (43.57 ± 4.52), DND42 > Rab11, KG02457/AP2σang7AP2σ (38.03 ± 3.60), and WTD42 > Rab11, KG02457/AP2σang7AP2σ (32.92 ± 2.14). Error bars represent SEM; statistical analysis is based on one-way ANOVA followed by post hoc Tukey’s multiple-comparison test. **p < 0.01; ns, not significant. D, Quantification of average mEJP amplitude in control (0.56 ± 0.01), DND42 > Rab11 (0.96 ± 0.08), WTD42 > Rab11 (0.78 ± 0.05), heteroallelic KG02457/AP2σang7AP2σ (1.19 ± 0.05), DND42 > Rab11, KG02457/AP2σang7AP2σ (0.13 ± 0.14), and WTD42 > Rab11, KG02457/AP2σang7AP2σ (1.46 ± 0.13). Error bars represent SEM; statistical analysis is based on one-way ANOVA followed by post hoc Tukey’s multiple-comparison test. **p < 0.01; ns, not significant. E, Histograms showing average mEJP frequency in control (1.38 ± 0.10), DND42 > Rab11 (4.17 ± 0.47), WTD42 > Rab11 (3.78 ± 0.31), heteroallelic KG02457/AP2σang7AP2σ (5.01 ± 0.23), DND42 > Rab11, KG02457/AP2σang7AP2σ (4.33 ± 0.23), and WTD42 > Rab11, KG02457/AP2σang7AP2σ (4.06 ± 0.34). Error bars represent SEM; statistical analysis is based on one-way ANOVA followed by post hoc Tukey’s multiple-comparison test. **p < 0.01; ns, not significant. F, Quantification of quantal content in control (50.56 ± 5.82), DND42 > Rab11 (53.14 ± 4.91), WTD42 > Rab11 (66.11 ± 5.48), heteroallelic KG02457/AP2σang7AP2σ (38.95 ± 3.65), DND42 > Rab11, KG02457/AP2σang7AP2σ (30.14 ± 3.03), and WTD42 > Rab11, KG02457/AP2σang7AP2σ (25.07 ± 0.93). Error bars represent SEM; statistical analysis is based on one-way ANOVA followed by post hoc Tukey’s multiple-comparison test. **p < 0.01; ns, not significant. Download Figure 8-3, TIF file.Overexpression of Rab11 does not restore the functional defects of 10.1523/ENEURO.0044-22.2022.f8-4Extended Data Figure 8-4σ2-adaptin mutants. A, Representative traces of mEJP in control, CAD42-Gal4 > Rab11, heteroallelic KG02457/AP2σang7AP2σ, and CA, AP2σang7/AP2σKG02457D42-Gal4 > Rab11 larvae. B, Representative traces of EJP in control (1118w), CAD42-Gal4 > Rab11, heteroallelic KG02457/AP2σang7AP2σ, and CA, AP2σang7/AP2σKG02457D42-Gal4 > Rab11 larvae. C, Quantification of average mEJP amplitude (mV) in control (0.67 ± 0.04), CAD42-Gal4 > Rab11(1.13 ± 0.17), heteroallelic KG02457/AP2σang7AP2σ (1.05 ± 0.09) and CA, AP2σang7/AP2σKG02457D42-Gal4 > Rab11 (1.30 ± 0.13). Error bars represent SEM; statistical analysis is based on one-way ANOVA followed by post hoc Tukey’s multiple-comparison test. **p < 0.01; ns, not significant. D, Quantification of average EJP amplitude in control (44.52 ± 1.07), CAD42-Gal4 > Rab11 (56.85 ± 4.221), heteroallelic KG02457/AP2σang7AP2σ (35.40 ± 1.925) and CA, AP2σang7/AP2σKG02457D42-Gal4 > Rab11 (41.41 ± 1.340). The error bars represent the SEM; statistical analysis is based on one-way ANOVA followed by post hoc Tukey’s multiple-comparison test. **p < 0.01; ns, not significant. E, Histograms showing quantal content in the control (63.43 ± 4.81), CAD42-Gal4 > Rab11(26.88 ± 4.42), heteroallelic KG02457/AP2σang7AP2σ (47.08 ± 4.13) and CA, AP2σang7/AP2σKG02457D42-Gal4 > Rab11 (37.38 ± 5.83). The error bars represent the SEM; statistical analysis is based on one-way ANOVA followed by post hoc Tukey’s multiple comparison test. **p < 0.01; ns, not significant. Download Figure 8-4, TIF file.Constitutively active Rab11 does not restore the physiological defects of σ2-adaptin and Rab11 increase Tkv accumulation and BMP signaling to generate similar NMJ phenotypes via independent pathways.In conclusion, Endo, Synj, nwk, shi, Clc, brat, and σ2-adaptin mutant NMJs  are reduced by half in σ2-adaptin mutant synapses.BMP signaling has been extensively studied in the context of neuronal growth in which the activated Tkv is endocytosed and fuse with the early endosomes, where it activates downstream signaling molecules. The signaling is attenuated when these activated receptor-containing vesicles recycle back to the plasma membrane or fuse with lysosomes for degradation . Trafficσ2-adaptin mutant synapses. The intensity profile of Tkv and HRP across the bouton shows that σ2-adaptin mutant has a higher intensity of Tkv at the membrane, indicating that a significant proportion of the Tkv receptors are accumulated at the presynaptic membrane. The pattern of Tkv enrichment in σ2-adaptin and Rab11 mutants were distinct. While σ2-adaptin mutant synapses showed Tkv enriched at synaptic membranes, Rab11 mutants had a rather punctate distribution within the bouton. The differential distribution of these proteins could be because of their distinct roles in the neurons. While σ2-adaptin plays a critical role in retrieving the SV-membrane from the presynaptic membrane, Rab11 is a component of the endolysosomal machinery. A clear separation of Tkv signals/pixels distinguishing plasma membrane Tkv from endosomal Tkv is challenging at the NMJ, given the resolution limit of the confocal system. The difference in Tkv distribution in σ2-adaptin and Rab11 mutants also argues for additional pathways, other than Rab11, through which σ2-adaptin may regulate BMP signaling.Interestingly, levels of early and late endosomes marked with Rab5 and Rab7, respectively, remain unaffected at the σ2-adaptin mutants could be accumulated either at the plasma membrane because of inefficient CME or at the early endosomes caused by inefficient recycling. If Tkv were accumulated at the early endosomes, we would expect greater colocalization with Rab5, which is not the case. However, based on reduced Rab11 staining at the σ2-adaptin mutant synapses, we conclude that the portion of the receptors in Rab5 positive early endosomes fail to recycle back to the plasma membrane. This conclusion also fits with previous observations that defective CME results in the accumulation of endosome-like structures in cultured hippocampal neurons (Drosophila NMJ, endocytosis attenuates BMP signaling (Drosophila wing discs and intestinal stem cells, endocytosis facilitates the signaling cascade by internalizing Tkv (σ2-adaptin mutants fail to recycle remaining receptors from early endosomes leading to enhanced signaling and NMJ growth defects.Tkv receptors in  neurons  and is s neurons . At the ignaling , whereaszing Tkv , pointinwit mutants, the size of the NMJ is significantly reduced with concomitant reduced evoked excitatory potentials (tkv and sax, co-Smad Medea, and transcription factor Mad showed that these NMJs have smaller synapses with severe functional deficits (gbb mutant larvae also exhibit shorter NMJs with severely reduced evoked potentials (σ2-adaptin mutant synapses show a modest reduction in evoked potentials, and the protein is dispensable for maintaining basal synaptic transmission (Endo, Synj, and Dap160, show a rapid stimulus-dependent decline in EJP amplitude that recovers following a period of rest after the high-frequency stimulation paradigm (σ2-adaptin mutants do not recover from synaptic depression even after the 90s rest after the cessation of high-frequency stimulation (1118wcontrols. EJP and high-frequency recordings from σ2-adaptin mutant synapses with one copy of 7tkv did not show any rescue in synaptic function. These data drive the conclusion that partial reduction of BMP pathway components can only rescue morphologic defects in σ2-adaptin mutants but not functional aspects and that morphologic and functional deficits can be discriminated in these mutants. Besides, the partial rescue of bouton size and bouton clustering at the NMJ argues for possible deregulation of multiple signaling pathways in σ2-adaptin mutants that remain to be explored.Morphologic features of synapses often dictate functional outcomes, and physiological analyses of BMP signaling mutants reveal the same. In tentials . Analysedeficits . Consisttentials . σ2-adapsmission . Howeverparadigm . In our mulation . This obDrosophila NMJ. First, we show using genetics that the morphologic defects in σ2-adaptin mutant synapses can be partially rescued by introducing a mutant copy of the BMP receptors, tkv, and wit. We also show a direct epistatic interaction between σ2-adaptin and the inhibitory Smad, Dad. Second, using immunohistochemistry, we show that σ2-adaptin mutant synapses accumulate Tkv at the plasma membrane and some of these receptors that are endocytosed and make it to the early endosomes fail to recycle back to the plasma membrane. Third, our electrophysiology data establish that morphologic and functional defects can be discriminated in σ2-adaptin mutants. Finally, our electron micrographs provide evidence for the presence of large endosomes and support our conclusion that σ2-adaptin is critically required at a later step of vesicle regeneration following endocytosis from the plasma membrane.Our study uncovers and extends the existing knowledge of synaptic growth signaling and endocytosis. We provide four lines of evidence on the critical role of σ2-adaptin in modulating BMP-dependent synaptic growth signaling at the σ2-adaptin mutant synapses that could be restored by neuronal expression of σ2-adaptin transgenes. However, the structural and functional defects of σ2-adaptin mutants could not be restored by neuronal expression of Rab11WT or Rab11CA. This rules out the notion that σ2-adaptin phenotypes result from the observed reduction of Rab11. The Rab11 mutant used in this study is a hypomorph with substantial Rab11 protein being detected by us and others (Rab11 hypomorphic mutants may explain why pMad levels were not as upregulated when compared with σ2-adaptin mutants. Finally, the separation of Tkv immunoreactivity from the synaptic membrane versus that of the endosomal membrane is challenging, given the resolution of the imaging system used here. Our electron micrographs, however, convincingly show an accumulation of endosome-like structures close to the plasma membrane in σ2-adaptin mutant boutons. The endosome-like structures observed in the σ2-adaptin mutants are strikingly similar to structures previously reported for clathrin (While this study does not report a direct biochemical or epistatic interaction between σ2-adaptin and Rab11, we observed a significant reduction in Rab11 immunoreactivity in d others . This prclathrin , AP180 (clathrin , and Rabclathrin , among oDrosophila NMJ. In the absence of AP2, recycling of Tkv is compromised, which results in its enrichment at the presynaptic membrane and/or in the early endosomes leading to elevated BMP signaling and synaptic overgrowth (We propose a model in which σ2-adaptin/AP2-complex is required for the attenuation of BMP signaling at the ergrowth . This st


This project aims to develop and evaluate a method for accurately determining time-integrated activities (TIAs) in single-time-point (STP) dosimetry for molecular radiotherapy. It performs a model selection (MS) within the framework of the nonlinear mixed-effects (NLME) model (MS–NLME).111In]In-DOTATATE activity in kidneys at T1 = (2.9 ± 0.6) h, T2 = (4.6 ± 0.4) h, T3 = (22.8 ± 1.6) h, T4 = (46.7 ± 1.7) h, and T5 = (70.9 ± 1.0) h post injection were obtained from eight patients using planar imaging. Eleven functions were derived from various parameterisations of mono-, bi-, and tri-exponential functions. The functions’ fixed and random effects parameters were fitted simultaneously (in the NLME framework) to the biokinetic data of all patients. The Akaike weights were used to select the fit function most supported by the data. The relative deviations (RD) and the root-mean-square error (RMSE) of the calculated TIAs for the STP dosimetry at T3 = (22.8 ± 1.6) h and T4 = (46.7 ± 1.7) h p.i. were determined for all functions passing the goodness-of-fit test.Biokinetic data of [NLME–PBMS and TIA3-parameters were 7.8% and 10.9% (for STP at T3), and 4.9% and 10.7% (for STP at T4), respectively.The function 111In-DOTATATE data, showing that four-parameter functions perform better than three- and five-parameter functions.An MS–NLME method was developed to determine the best fit function for calculating TIAs in STP dosimetry for a given radiopharmaceutical, organ, and patient population. The proof of concept was demonstrated for biokinetic The online version contains supplementary material available at 10.1186/s40658-023-00530-1. Individual dosimetry in molecular radiotherapy (MRT) is often not performed in the clinic, as it requires sequential imaging to determine the time-integrated activity (TIA) –4. These177Lu]Lu-PSMA therapy [177Lu]Lu-DOTATATE in kidneys during PRRT using a bi-exponential function and single-time-point (STP) imaging with SPECT/CT In-DOTATATE was administered intravenously to the patients as a (51 ± 8) min infusion. Planar whole-body scintigraphies using a double-head gamma camera  were performed at T1 = (2.9 ± 0.6) h, T2 = (4.6 ± 0.4) h, T3 = (22.8 ± 1.6) h, T4 = (46.7 ± 1.7) and T5 = (70.9 ± 1.0) h p.i. In-DOTATATE were used as a surrogate for predicting the kinetics of [90Y]Y-DOTATATE used for peptide-radionuclide therapy, as suggested in the literature In-DOTATATE in kidneys  functions with 3, 4, and 5 parameters and different parameterisations were used to fit the biokinetics of 11InIn-DO0-4min-1 ), \documm et al. , existinm et al. , 25 and Parameters in the NLME model consist of the fixed and random effects Eqs. –13 as re in Eqs.  were mod111In]In-DOTATATE in kidneys  and T4 = (46.7 ± 1.7) p.i. were used for the STP fitting as suggested in the literature In-DOTATATE were used for the proof of concept of STP approach with NLME modelling plus a model selection for the fit function. Biokinetic data of [111In]In-DOTATATE can be used as a surrogate for predicting the kinetics of [90Y]Y-DOTATATE In-DOTATATE as proof of concept: Since STP dosimetry depends on the fit function used, determining the best fit function is essential for an optimal STP dosimetry method. In general, NLME modelling, a standard procedure in pharmacokinetic science, is a promising approach to individualise MRT dosimetry by STP measurements.To determine the best fitting function for calculating TIAs in STP dosimetry for a given radiopharmaceutical, organ and patient population, we proposed a population-based model selection method (MS–NLME). The application of this method was demonstrated for the biokinetics of [Additional file 1. Time-activity curves, relative deviations, and root-mean-square error of sum of exponentials functions.


The indenoquinoxaline ring is almost in the same plane (r.m.s. deviation = 0.0181 Å).In the title compound, C The packing is shown in Fig. 2The molecular structure of the title compound is shown in Fig. 1H-indenoquinoxaline; Chen et al., 2020b]quinoxaline ring (r.m.s. deviations = 0.1197 Å) is twisted with respect to the two benzene ring systems by 70.0 (4) and 67.6 (3)°, respectively.One similar structure has been reported previously  and 4,5-dimethylbenzene-1,2-diamine  in ethanol (100 ml) was heated to reflux under stirring for 5 h. 7,8-Dimethyl-11H-indenoquinoxalin-11-one was obtained as a yellow powder by filtering after cooling, yield 82%. Single crystals of the title compound suitable for X-ray analysis were obtained by slow evaporation of a methanol solution.A mixture of 1Crystal data, data collection and structure refinement details are summarized in Table 110.1107/S2414314621000183/xu4042sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2414314621000183/xu4042Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2414314621000183/xu4042Isup3.cmlSupporting information file. DOI: 2054191CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Convolvulus scammonia by setting mass rang and mass defect range from high-resolution MS data. The full-scan mass data were acquired by high-performance liquid chromatography coupled with Q Exactive Plus hybrid quadrupole-orbitrap mass spectrometer that featured high resolution, mass accuracy, and sensitivity. To screen resin glycosides, three parent filter m/z 871, m/z 853, and m/z 869 combined with diagnostic fragment ions (DFIs) approach were applied to remove the interference from complex herbal extract. The targeted components were characterized based on detailed fragment ions. Using this approach, 80 targeted components, including 22 glycosidic acids and 58 resin glycosides were tentatively identified. The present results suggested that the proposed MDF strategy would be adaptable to the analysis of complex system in relevant filed.This work aimed to develop and evaluate a post-acquisition data processing strategy, referred to as a mass defect filter (MDF), for rapid target the resin glycosides in root of  Convolvulus scammonia belongs to morning glory family (Convolvulaceae) and its extractive scammony, known commonly as resin glycosides which are unique secondary metabolites in natural. These active principles are responsible for the drastic purgative action and Convolvulaceous plants used in traditional medicine throughout the world, especially Convolvulus scammonia −, m/z 593 [739 − C6H10O4]− combined with m/z 447 [593 − C6H10O4]− were checked. Characteristic ion m/z 285 [447 − C6H10O5]−, which was 14 Da higher than jalapinolic acid [(S)-11-hydroxyhexadecanoic acid] indicated this resin glycoside having a hydroxylated C17 fatty acid as its aglycone.Turpethic acids C , a glyco− at m/z 1265  from m/z 1265 [M − H − C5H10O2]−, successively loss of 2-methylbutyric acid and tiglic acid  to produce m/z 1063 [1265 − C5H8O2 − C5H10O2]−, m/z 1061 [1265 − C5H10O2 − C5H10O2]−, m/z 1019 [1265 − C11H18O6 (O-methylbutyrylglucose unit)]−m/z 979 [1265 + H2O − C5H10O2 − C5H10O2 − C5H8O2]−, m/z 961 [1265 − C5H10O2 − C5H10O2 − C5H8O2]− together with diagnostic fragment ion m/z 835 [1265 + H2O − C11H18O6 − C5H8O2 − C5H10O2]−, m/z 579 [835 − 146 − (146 − 2 × H2O)]−, m/z 561 [835 − 146 − (146 − H2O)]−, m/z 543 [835 − 146 − 146]−, m/z 433 [579 − 146]−, m/z 417 [579 − 162]− as well as m/z 271 [417 − 146]− were observed.Turpethoside B exhibited [M − H]m/z 1265 , fragmenm/z 871 (C40H71O20) picked as filter for glycosidic acids, m/z 853 (C40H69O19) and m/z 869(C40H69O20) for resin glycosides. Transformations such as 2-methylbutyric acid (Mba), tiglic acid (Tga), isobutyric acid (Iba), -3-hydroxy-2-methylbutyric (Nia), 3-hydroxy-methylenebutyric (Hma) were supplied to MDF workflow. Using the mass defect values calculated above and the exact mass values calculated from the elemental composition input -11-hydroxyhexadecanoic acid] is the most frequently aglycone for the macrocyclic lipooligosaccharide core. Therefore, the parent drug filter was based on the jalapinolic acid, on input , mass toon input . The usem/z 1170.60286 at 59.46 min was largely improved.The generated chromatograms are displayed in m/z 257, m/z 271, m/z 285 indicated the presence of the hydroxylated fatty acid with chain lengths of C15, C16, and C17, respectively. A total of 22 glycosidic acids were detected and tentatively characterized in root of Convolvulus scammonia, in which 21 compounds were based on scammonic acid A parent filter, Their ESI-MSn information is shown in m/z 1170 was founded that scammonic acid B as its parent drug. Diagnostic fragment ion at m/z 271 was the common aglycone, suggesting the presence of jalapinolic acid.Glycosidic acids exhibited deprotonated ions as the base peak were observed; neutral loss of monosaccharide units including deoxyhexose, hexose and short organic acid moieties such as tiglic acid, 2-methylbutyric acid, -3-hydroxy-2-methylbutyric were the main fragment patterns; aglycone ions m/z 1053,the HCD-MS2 of which exhibited produced ions at m/z 953 [M − H − 100(3hydroxy-2-methylbutyric acid residue)]−, m/z 871 [M − H − 100(3-hydroxy-2-methylbutyric acid residue) − 82(tiglic acid residue)]−, which indicated the parent drug filter was scammonia acid A, diagnostic fragment ions m/z 853 [1053 + H2O − 118 − 100]−, m/z 835 were generated due to successively loss of 3-hydroxy-2-methylbutyric acid and tiglic acid from m/z 1053, furthermore, successive elimination of deoxyhexose moiety (146 Da), hexose moiety (162 Da) to generate m/z 725 [871 − 146]−, m/z 579 [725 − 146 − 146]−, m/z 417 [579 − 162]− coupled with m/z 271 [M − H − 100 − 82 − 146 − 146 − 162 − 146]− supported the existence of scammonic acid A. It was conclusion that peak 1, 5, 8 were isomer of 3-hydroxy-2-methylbutyric acid + tiglic acid + scammonic acid A.Peak 1, 5, 8 all gave high intensity of deprotonated ion at − ion at m/z 1055, which was tentatively identified as isomer of 3-hydroxy-2-methylbutyric acid + 2-methylbutyric acid + scammonic acid A. The typical fragment ions m/z 955 [M − H − 100]−, m/z 871 [M − H − 100 − 84(2-methylbutyric acid residue)]−, m/z 853 [M − H + H2O − 118 − 102]− represented successively loss of 3-hydroxy-2-methylbutyric acid residue (100 Da), 2-methylbutyric acid residue (84 Da) from deprotonated ion, respectively. It’s also gave ions at m/z 579 , m/z 417  and m/z 271 in MS/MS spectrum by cleavage of glycosidic bond on m/z 871(scammonic acid A).Peak 2, 6 had the same [M − H]m/z 1169, its diagnostic ions m/z 887 of scammonic acid B can distinguished itself from scammonic acid A (m/z 871). Fragment ions at m/z 969 [1169 − 100(3-hydroxy-2-methylbutyric acid residue) − 100]−, m/z 951 [1169 − 100 − 82(tiglic acid residue)]−, m/z 887 [969 − 82]− were attributed to loss of C5H6O residue  from m/z 969, m/z 851 [951 − 100]− were also detected. Characteristic ions m/z 579 were observed for elimination of deoxyhexose moiety 146 Da and hexose moiety 162 Da from scammonic acid B (m/z 887), in addition, m/z 417 [579 − 162]− combined with m/z 271 [417 − 146]− supported that oligoglycosides conjunction type in scammonic acid B was deoxyhexose–hexose–deoxyhexose–hexose. According to information mentioned above, peak 3 was tentatively characterized as 3-hydroxy-2-methylbutyric acid + 3-hydroxy-2-methylbutyric acid + tiglic acid + scammonic acid B.Peak 3 was eluted at tR 59.405 min with precursor ion at m/z 1071 with elemental composition C50H87O24, m/z 971 indicated the transformation was 3-hydroxy-2-methylbutyric acid residue [M − H − 100]−, scammonic acid A as parent drug template was supported from diagnostic ions m/z 871 [971 − 100]−, m/z 853 [1071 + H2O −118 − 118]−, m/z 725 [871 − 146(deoxyhexose moiety)]−, m/z 579, m/z 561 and m/z 417. It supposed that peak 4 was 2 mol 3-hydroxy-2-methylbutyric acid substituted on scammonic acid A.Peak 4 had high intensity ion at m/z 1041 eluted at retention time of 65.234, fragmentation ions at m/z 941 which was attributed to loss of 3-hydroxy-2-methylbutyric acid residue (100 Da) from m/z 1041, m/z 923 [1041 − 118]−. Typical ions at m/z 871 [M − H − 100(3-hydroxy-2-methylbutyric acid resiue) − 70(isobutyric acid residue)]−, m/z 853 [1041 + H2O − 118 − 88]−, m/z 835 [1041 − 118 − 88]− as evidence for the presence of scammonic acid A. In addition, m/z 579, m/z 417 as well as m/z 271 supported that. The tentative identification given to peak 7 was 3-hydroxy-2-methylbutyric acid + isobutyric acid + scammonic acid A.Peak 7 gave deprotonated ion at 55H94O25 . Fragmentation ions at m/z 1053 [M − H − 100]−, m/z 953 [M − H − 100 − 100]− demonstrated that the existence of 3-hydroxy-2-methylbutyric acid, successive loss of tiglic acid to give fragmentation ion at m/z 871 which was diagnostic ion of scammonic acid A. furthermore, m/z 579, m/z 417, m/z 217 were founded in MS/MS spectrum.Peak 9, 10, 11, 12, 13 were tentatively characterized as isomer of 3-hydroxy-2-methylbutyric acid + 3-hydroxy-2-methylbutyric acid + tiglic acid + scammonic acid A according to elemental composition Cm/z 1123, which was 100 Da higher than m/z 1023, it indicated that the presence of 3-hydroxy-2-methylbutyric acid. In addition to this, there were similarity fragmentation ions with m/z 1023 [M − H − 100(3-hydroxy-2-methylbutyric acid residue)]−, such as m/z 941, m/z 871, m/z 579, m/z 417, m/z 271. Above all, we inferred that ion at m/z 1123 was based on parent filter scammonic acid A and substituted by 3-hydroxy-2-methylbutyric acid, tiglic acid as well as isobutyric acid.Peak 14, 16 as well as 19 displayed the same deprotonated ion at − ions at m/z 1023 with the elemental composition of C49H84O22. In the MS/MS spectrum, m/z 941 indicated that loss of substitutes tiglic acid residue (82 Da) from [M − H]−, and then successively loss of isobutyric acid residue (70 Da) to produce ions at m/z 871, fragmentation ions m/z 725, m/z 579 suggested successive loss of rhamnose monohydrate from m/z 871, neutral loss of glucose (162 Da) to produce ion at m/z 417. Diagnostic ion m/z 271 supplied the evidence for the existence of jalapinolic acid aglycone. We inferred from fragmentation ions that peak 15 was scammonic acid A combined with 1 mol each of tiglic acid and isobutyric acid.Peak 15 showed [M − H]− ion at m/z 1137. Prominent ion at m/z 871 [1137 − 82(tiglic acid residue) − 100(3-hydroxy-2-methylbutyric acid residue − 84(2-methylbutyric acid residue)]− provided evidence for the existence of scammonic acid A. Fragmentation ions at m/z 1055, m/z 955, m/z 937 [1137 + H2O − 100 − 118]−, m/z 835 [1137 + H2O − 100 − 118 − 102]− were checked. Breakage of glycosidic bond was the main fragmentation pathway of scammonic acid A, from which m/z 579 [871 − 146 − 146]−, m/z 417 [871 − 146 − 146 − 162]− and m/z 217 [871 − 146 − 146 − 162 − 146]− were generated. We presumed that peak 17 and 22 were isomer of scammonic acid A + tiglic acid + 3-hydroxy-2-methylbutyric acid + 2-methylbutyric acid.Peak 17 and 22 exhibited [M − H]−m/z 1037 with formula C50H86O22 eluted at 74.561 min, in analysis of its MS/MS spectrum, the [M − H − 82(tiglic acid residue)]− ion at m/z 955, m/z 935 [M − H − 102(2-methylbutyric acid)]−, [M − H − 82 − 84(2-methylbutyric acid resiude)]− ion at m/z 871 and characteristic ions of scammonic acid A were checked. Compared with peak 18, 23, peak 19 was 100 Da lower, it illustrated that there was absence of 3-hydroxy-2-methylbutyric acid. Therefore, we inferred that peak 18 was based on scammonic acid A and substituted by tiglic acid and 2-methylbutyric acid.Peak 18, the [M − H]− at m/z 1039, fragmentation ions at m/z 955, m/z 871 demonstrated that 2 mol of 2-methylbutyric acid substituted on scammonic acid A, m/z 579, m/z 417 combined with m/z 271 as notable markers for scammonic acid A were checked in MS/MS spectrum. Peak 20 was tentatively characterized as scammonic acid A + 2-methylbutyric acid + 2-methylbutyric acid.Peak 20 showed [M − H]m/z 1125 was eluted at 78.93min. Study of the MS/MS spectrum of glycosidic acids, loss of organic acid and cleavage of glycosidic bond were main fragmentation pathway, ions at m/z 1025 [M − H − 100]−, m/z 941 [M − H − 100 − 84]− as well as m/z 871 [M − H − 100 − 84 − 70]− were generated according to successively loss of 3-hydroxy-2-methylbutyric acid, 2-methylbutyric acid and isobutyric acid from deprotonated ion m/z 1125. Diagnostic ions of scammonia acid A at m/z 579, m/z 417, m/z 271 were observed. We presumed that peak 21 was scammonic acid A + 3-hydroxy-2-methylbutyric acid + 2-methylbutyric acid and isobutyric acid.Peak 21 with high intensity at Convolvulusscammonia L share the common structure of a macrolactone composed by one acylated glycosidic acid. Through the post-data mining strategy MDF, totally 58 resin glycosides were checked, in which 56 resin glycosides were established on parent filter m/z 853 except ion at m/z 1151 and m/z 1149.Resin glycosides in 56H94O20. The precursor ion [M − H]−m/z 1149 firstly elimination of C2H4O (44 Da), fragmentation ion m/z 1049 was attributed to loss of 100(3-hydroxy-2-methylbutyric acid residue) from precursor ion. Ion at m/z 949 [1049 − 100]− indicated there was existence of 2 mol 3-hydroxy-2-methylbutyric acid in the structure. Diagnostic fragment ion m/z 867 [1049 − 100 − 82(tiglic acid residue)]− coupled with noteworthy ions m/z 849, m/z 593 [867 − 146 − (146 − H2O)]−, m/z 575 [867 − 146 − 146]−, m/z 431 [593 − 162]−, m/z 413 [575 − 162]− as well as m/z 285 [431 − 146]− were checked. These ions showed the evidence for the existence of C17 fatty acid aglycone. According to the detailed fragmentation ion information and fragment rules, we inferred that compound with structural of m/z 867 + 2 mol 3-hydroxy-2-methylbutyric acid + tiglic acid.Peak was eluted at 85.271min with elemental composition C55H92O25 (m/z 1151) was eluted at 71.689 min. In MS/MS spectrum [M + HCOO]− ion at m/z 1197 was observed, its deprotonated ion at m/z 1151 successively loss of 3-hydroxy-2-methylbutyric acid (118 Da) to generate ion at m/z 1051 [M − H − 100(3-hydroxy-2-methylbutyric acid residue)]− and m/z 951 [M − H − 100]−, respectively. Fragmentation ion m/z 1033 [1151 − 118]−, 1007 [1151 − 144(hexose unit − H2O)]−, m/z 933 [1151 + H2O − 118 − 118]− coupled with m/z 907 [1151 − C11H16O6(O-tigloylhexose unit)]− and m/z 905 [1151 − C11H18O6(O-3-hydroxy-2-methylbutyrylpentose unit)]− were checked. Diagnostic ion at m/z 869 [M − H − 100 − 100 − 82(tiglic acid residue)]− demonstrated the sugar units were hexose–pentose–pentose–hexose which linked to jalapinolic acid to form macrolactone ring, it also illustrated the existence of tiglic acid. According to the fragmentation ions, we inferred this compound was based on parent drug filter m/z 869 and substituted by 2 mol 3-hydroxy-2-methylbutyric acid and 1 mol tiglic acid.Peak with elemental composition Cm/z 853, totally 56 peaks were tentatively characterized, the detail of ESI-MSn information is shown in −, [M + Cl]− combined with [M − H]− were observed, it is different from glycosidic acids;adduct ion [M + HCOO]loss of short organic acid such as 2-methylbutyric acid, tiglic acid, isobutyric acid, -3-hydroxy-2-methylbutyric, 3-hydroxy-2-methylenebutyric were common characteristics;breakage of glycosidic linkage is prone to loss of 162 Da, 146 Da;2H4O (44 Da) was observed when -3-hydroxy-2-methylbutyric substituted on resin glycosidesloss of Cm/z 853 with lower intensity was obtained and identical ions at m/z 579, m/z 561, m/z 543, m/z 399, m/z 417 combined with m/z 271 were discovered.parent drug filter ion at Peaks listed below were all based on parent template m/z 937 and adduct ion [M + HCOO]− at m/z 983, [M + Cl]− at m/z 973 were found. Diagnostic ion at m/z 853 [M − H − 84(2-methylbutyric acid residue)]−, m/z 835 [937 − 102]− were discovered in MS/MS spectrum, from which we inferred that there was 2-methylbutyric acid substituted on parent drug filter m/z 853. Therefore, Peak 1′ was tentatively identified as m/z 853 + 2-methylbutyric acid.Peak 1′ displayed precursor ion at − at m/z 1081 with high intensity and [M + Cl]− at m/z 1071 in MS spectrum, analysis of MS/MS spectrum, [M − H]− at m/z 1035 (C50H83O22) gave high abundance, fragmentation ion at m/z 991 [M − H − 44]−, m/z 953 [1035 − 82(tiglic acid residue)]− and m/z 935 [1035 − 100(3-hydroxy-2-methylbutyric)]− with highest intensity appeared. Diagnostic ion m/z 853 [935 − 82(tiglic aid residue)]− with ion m/z 835 [1035 + H2O − 118 − 100]− served as evidence for parent drug. Based on fragmentation rules, peak 2′, 5′, 6′, 8′, 11′, 13′, 22′ were characterized as isomer of m/z 853 + 3-hydroxy-2-methylbutyric + tiglic aid.Peak 2′, 5′, 6′, 8′, 11′, 13′, 22′ all exhibited [M + HCOO]− at m/z 1069 and [M − H]− at m/z 1023 in MS/MS spectrum. Ion at m/z 979 indicated loss of 44 Da (C2H4O) from deprotonated ion, m/z 935 through loss of isobutyric acid from m/z 1023, m/z 923 [M − H − 100]− demonstrated the presence of -3-hydroxy-2-methylbutyric, noteworthy ion at m/z 835 [M − H − 100 − 88]− as parent drug filter observed. A tentative identification of Peak 3′ was m/z 853 + -3-hydroxy-2-methylbutyric + isobutyric acid.Peak 3′ showed [M + HCOO]− at m/z 1123 was 100 Da higher than m/z 1023, which had the same fragmentation pattern with m/z 1023, namely loss of organic acid and breakage of glycosidic linkage. From fragmentation ions m/z 1079, m/z 1035, m/z 1023 as well as m/z 835, we presumed the structure was m/z 853 + 2 mol 3-hydroxy-2-methylbutyric + isobutyric acid.Peak 4′, 20′, 25′ all gave [M − H]− at m/z 1083 and [M + Cl]− at m/z 1073 in MS spectrum, analysis of MS/MS spectrum [M − H]− at m/z 1037 combined with m/z 993 [M − H − 44]−, m/z 937 [M − H − 100(3-hydroxy-2-methylbutyric residue)]− as well as markable fragmentation ion at m/z 853 [937 − 84]−, m/z 579, m/z 561, m/z 417, m/z 217 were checked. Peak 7′, 10′, 16′, 18′ were tentatively identified as isomer of m/z 853 + 3-hydroxy-2-methylbutyric + 2-methylbutyric acid.Peak 7′, 10′, 16′, 18′ all exhibited [M + HCOO]− at m/z 1099 with high abundance. In analysis of MS/MS spectrum, [M − H]− at m/z 1053 with elemental composition C50H85O23 was prone to loss of 44 Da to form ion at m/z 1009, ions at m/z 909 and m/z 891 were formed from [M − H − 44 − 100(3-hydroxy-2-methylbutyric residue)]−, [M − H − 44 − 118]−, respectively. Fragmentation ions at m/z 953 [M − H − 100]−, m/z 853 [M − H − 100 − 100]−, m/z 835 [1053 + H2O − 118 − 118]− both of these fragmentation ions illustrated the existence of 2 mol 3-hydroxy-2-methylbutyric. According to the fragment rules, peak 9′ and 14′ were tentatively characterized as isomer of m/z 853 + 2 mol 3-hydroxy-2-methylbutyric.Peak 9′, 14′ showed [M + HCOO]m/z 853 + 2 mol 3-hydroxy-2-methylbutyric + tiglic acid according to fragmentation rule of resin glycosides. The [M + HCOO]− ion at m/z 1181 with high intensity and [M + Cl]− ion at m/z 1171 were observed in MS spectrum, in analysis of its MS/MS spectrum, the noteworthy ions m/z 1091 and m/z 1047 were due to successively loss of C2H4O (44 Da) from precursor ion m/z 1135, ion at m/z 1035 was attributed to loss of 100 Da (3-hydroxy-2-methylbutyric residue) from [M − H]−, m/z 991 [1091 − 100]−, m/z 935 from [M − H − 100 − 100]−. Characteristic ion m/z 853 [M − H − 100 − 100 − 82]− was checked, it illustrated the existence of tiglic acid, diagnostic ions of at m/z 579, m/z 561, m/z 417 combined with m/z 217 supported the parent drug filter was m/z 853.Peak 12′, 15′, 17′, 19′, 21′, 23′, 26′, 28′, 41′ were tentatively identified as isomer of − at m/z 1137, successively loss of 44 Da from m/z 1137 to generate m/z 1093 and m/z 1049 with high abundance, according to fragmentation pattern of m/z 1135 we inferred there were 2 mol 3-hydroxy-2-methylbutyric substituted on resin glycosides. Ion at m/z 1037 −, m/z 961 [1061 − C5H8O2(tiglic acid)]−, m/z 917 [1005 − isobutyric acid]−, m/z 835 [917 − (tiglic acid − H2O)]− were found. Cleavage of glycoside bond was prone to produce diagnostic ions m/z 579, m/z 561, m/z 417 and m/z 271. We presumed that the structure of peak 27′, 30′, 36′, 42′, 46′, 54′ was 3-hydroxy-2-methylbutyric, tiglic acid combined with isobutyric acid substituted on parent drug m/z 853.Peak 27′, 30′, 36′, 42′, 46′, 54′ exhibited high intensity [M + HCOO]− at m/z 1153 and [M + Cl]− at m/z 1143. In the MS/MS spectrum [M − H]− at m/z 1107 with elemental composition C54H91O23 was observed, m/z 1005 was attributed to loss of 2-methylbutyric acid from 1107, m/z 989 [M − H − 118]− and m/z 905 [M − H − 102 − 100]− illustrated the presence of 3-hydroxy-2-methylbutyric. Cleavage of glycoside bond and loss of hexose moiety, pentose moiety as noteworthy fragment pathway was checked, the corresponding fragment ions at m/z 579 [835 − 146 − (146 − 2 × H2O)]−, m/z 561 [835 − 146 − (146 − H2O)]−, m/z 543 [835 − 146 − 146]−, m/z 399 [561 − 162]−, m/z 417 [579 − 162]− combined with m/z 271 [417 − 146]− were discovered. Peak 31′, 48′, 51′ were tentatively characterized as isomer of m/z 853 + 2-methylbutyric acid + 3-hydroxy-2-methylbutyric + isobutyric acid based on fragment rules.Peak 31′, 48′, 51′ displayed [M + HCOO]− at m/z 1053 and [M − H]− at m/z 1007 which was 100 Da lower than m/z 1107, it presumed absence of 3-hydroxy-2-methylbutyric. Fragment ion at m/z 923 [M − H − 84]−, m/z 905 [M − H − 102]− suggested the existence of 2-methylbutyric acid, ion at m/z 919 [M − H − 88]− coupled with m/z 835 [M − H − 102 − 70]− demonstrated the presence of isobutyric acid. The diagnostic ions m/z 579, m/z 561, m/z 417, m/z 399 as well as m/z 271 supported the breakage of glycosides bond from m/z 853. Therefore, the tentative identification given to peak 33′, 38′ was isomer of m/z 853 + 2-methylbutyric acid+ isobutyric acid.Peak 33′, 38′ gave [M + HCOO]m/z 853 + 2-methylbutyric acid + tiglic acid based on proposed fragment rules of resin glycosides. A high abundance of [M + HCOO]− ion at m/z 1065 coupled with [M + Cl]− at m/z 1054 were discovered. In analysis of MS/MS spectrum, the precursor ion at m/z 1019 gave high intensity, ion at m/z 937 [M − H − 82]−, m/z 919 [M − H − 100]− served as evidence for the existence of tiglic acid. Ion at m/z 853 [M − H − 82 − 84]− demonstrated the presence of 2-methylbutyric acid.Peak 35′, 40′ were tentatively characterized as isomer of − at m/z 1119, a difference of 100 Da between m/z 1019 and m/z 1119 suggested 3-hydroxy-2-methylbutyric substituted on resin glycoside, ion at m/z 1001 [M − H − 118]− and m/z 1019 [M − H − 3-hydroxy-2-methylbutyric residue]− supported that speculation. Ions at m/z 937 [1019 − 82]−, 917 [937 − 102]− coupled with m/z 835 were observed in MS/MS spectrum. According to fragment rules and information listed above, the tentative identification given to peak 37′, 49′, 52′, 56′ were isomer of m/z 853 + tiglic acid + 2-methylbutyric acid + 3-hydroxy-2-methylbutyric.Peak 37′, 49′, 52′, 56′ showed [M − H]− at m/z 1139 in MS spectrum, [M − H]− at m/z 1093 loss of C2H4O (44 Da) to form m/z 1049, product ions at m/z 1005 [M − H − 88]−, m/z 905 [M − H − 88 − 100]−, m/z 835 [M − H − 88 − 100 − 70]− were checked in MS/MS spectrum, we inferred peak 39′, 45′ were isomer of m/z 853 + 3-hydroxy-2-methylbutyric + 2 mol isobutyric acid.Peak 39′, 45′ exhibited [M + HCOO]− at m/z 1021, adduct ion [M + HCOO]− at m/z 1067, ion at m/z 937 [M − H − (102 − H2O)]− due to loss of 2-methylbutyric acid from deprotonated ion, m/z 919 [M − H − 102]− with high abundance were obtained. Characteristic ion m/z 853 was attribute to successively loss of 2-methylbutyric acid residue from m/z 1021, m/z 561, m/z 417, m/z 399 combined with m/z 271 were observed. Therefore, peak 43′, 47′ were tentatively characterized as isomer of m/z 853 + 2-methylbutyric acid+2-methylbutyric acid.Peak 43′, 47′ showed [M − H]m/z 1117, [M + HCOO]− ion at m/z 1163 was observed in MS spectrum. In MS/MS a predominant productive ion m/z 1017 [M − H − (118 − H2O)]−, coupled with m/z 999 [M − H − 118]−, m/z 917 [M − H − 118 − (100 − H2O)]− and m/z 935 [M − H − 100 − 82]−. Ion at m/z 835 combined with m/z 561, m/z 417, m/z 399, m/z 271 from the cleavage of glycoside bond were checked. We thus inferred the structure of peak 44′ and 50′ was 2 mol tiglic acid + 3-hydroxy-2-methylbutyric substituted on parent drug m/z 853.Peak 44′, 50′ exhibited deprotonated ion at − at m/z 1119 and its adduct ion [M + HCOO]− at m/z 1165, [M + Cl]− at m/z 1155 were found in MS spectrum. In analysis of its MS/MS spectrum, ion at m/z 1019 [M − H − C5H8O2]−, m/z 1001 [M − H − 118]− indicated the existence of 3-hydroxy-2-methylbutyric, m/z 919 [M − H − 118 − 82(100 − H2O)]−, m/z 835 [M − H − 118 − 82 − 84(102 − H2O)]− with high abundance were generated. According to proposed fragment pattern of resin glycoside, we gave tentative characterization to m/z 1119 was m/z 853 + 3-hydroxy-2-methylbutyric + tiglic acid + 2-methylbutyric acid.Peak 53′, 55′ were eluted at 91.147 min and 89.303 min, respectively. For structural elucidation, [M − H]Acetonitrile, methanol and formic acid (MS grade) were purchased from Fisher Scientific . Deionized water was obtained from Watson’s .References Turpethic acids C and Turpethoside B were provided by Professor Xiaoyi Wei, Key Laboratory of Plant Resources Conservation and Sustainable Utilization, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou, China.Convolvulusscammonia L. was provided by Xinjiang Uygur Medicine Limited Liability Company . The sample (1.0 g) was dispersed in 10 mL 100% methanol, and extraction was performed on a water bath at 25 °C assisted with ultrasound for 40 min. The extract was centrifuged at 14,000 rpm for 7 min, leading to the supernatant used as the test solution.The extract of ® HSS T3 column  maintained at 40 °C was used. The mobile phase consisted of acetonitrile (A) and 0.1% formic acid–water (B) with a gradient elution of 5% B at 0–1 min, 5–9% B at 1–4 min, 9–18% B at 4–22 min, 18–88% B at 22–92 min, 88–95% B at 92–92.1 min and 95% B at 92.1–110 min. A flow rate of 0.15 mL/min was set and injection volume was 1.00 μL.Chromatographic separation was performed on Ultimate 3000 RSLC system . An ACQ-UITY UPLCTM PLUS hybrid Quadrupole-Orbitrap Mass Spectrometer equipped with a heated ESI source  operating in the negative mode. The HESI parameters were set as follows: spray voltage, −2.8 kV; Capillary temperature 320 °C; sheath gas pressure, 35 arb; aux gas pressure, 10 arb; spare gas pressure, 0 arb; probe heater temperature, 350 °C; s-lens RF, 55 V. The Orbitrap analyzer scanned over m/z 100–1500 at a resolution of 70,000 in full scan MS1, and dynamic mass ranges in MS2 with resolution set at 13,500. AGC target values for MS1 and MS2 were 5e6 and 1e5, respectively. Maximum injection time (IT) for MS1 was defined as 100 ms and 50 ms for MS2.High-resolution MS data were recorded on a Q Exactive2 experiments were performed by high collision induced dissociation (HCD) to improve the sensitivity and offer more fragments information for structural elucidation at normalized collision energy (NCE) 10/30/50 V, with isolation window at 4.0 m/z. Dynamic exclusion allows each precursor ion to be selected 1 time before being excluded with 10 s duration. The HRMS full scan and HCD MS2 data were viewed and processed by Xcalibur 4.2. .MSResin glycosides analogues usually shared with the similar core structure; characteristic compounds were generated via various substituents. Therefore, with increasing the number of these substituted groups, the decimal mass and integer mass of the molecular weight would show the linear or fixed relationship in exploring the interested precursor ions. In other words, the elemental compositions of these compounds would produce regular changes with the increase of their molecular weights. Thus, their molecular ions and the isotope distributions can be dynamically presented by preferred mathematical method ,24,25,2640H72O20), MW 854 Da (C40H70O19), MW 870 Da (C40H70O20) were picked as parent drug filter, mass defect type was standard mass defect . Elemental composition prediction of the detected components was based on the following settings: elements in use, C 0–70, H 0–110, O 0–30; mass tolerance, <5 ppm; adduct species [M + HCOO]−, [M − H]−. The High-resolution MS data were processed by MDF using Compound Discovery 3.2 .The MDF strategy relies on the decimal mass shift to remove the interferences and to pick out the target ions for structural elucidation. Core substructure and different substituent combination were the two essential parts to realize the MDF approach. In the present study, Molecular Weight 872 Da -12-hydroxy-pentadecanoic acid was linked to C-2 of Rha to form a macrolactone ring, but the carboxyl group of jalapinolic acid was linked to C-3 of Rha to form a macrolactone ring as common appeared in Convolvulus scammonia, therefore, the abundance of dignositic fragment ions was a little bit different. In a word, DPIs analysis can provide a criterion to classify the target constituents detected into certain chemical families.In the present study, a sensitive and effective strategy for rapid identification of the characteristic structural analogues resin glycosides in cammonia ,11,12, tMass defect filtering-oriented identification of resin glycosides is definitely an effective strategy, there were more MDF methods, including multiple MDF, raster MDF, five-point screening MDF and polygonal MDF, developed for more accurate precursor ions filtered and different types of plant metabolites can be discriminated by sorts of MDF methods.


Moreover, we characterized the resulting monolithic integrated catalysts in detail and evaluated their catalytic activities for toluene combustion, in comparison to the controlled samples including only Pd NPs loading and the δ-MnO2 nanoflower arrays. Amongst all the monolithic samples, the Pd/δ-MnO2 nanoflower arrays monolithic catalyst with 0.05 wt% theoretical Pd loading delivered the best catalytic performance, reaching 90% toluene conversion at 221°C at a gas hourly space velocity (GHSV) of 10,000 h−1. Moreover, this sample displayed superior catalytic activity for o-xylene combustion under a GHSV of 10,000 h−1. The monolithic sample with optimal catalytic activity also displayed excellent catalytic stability after 30 h constant reaction at 210 and 221°C.Exploring high-efficiency and stable monolithic structured catalysts is vital for catalytic combustion of volatile organic compounds. Herein, we prepared a series of Pd/δ-MnO This optimal monolithic catalyst also exhibited an excellent long-term durability.Herein, by using a two-step synthetic route , δ-MnO2 10H14N2O8Na2·2H2O, EDTA), potassium permanganate (KMnO4), ammonium Chloride (NH4Cl), sodium hypophosphite (NaH2PO2·H2O), and ammonia water (25%) were bought from Guangzhou Chemical Reagent Co. Ltd. . Palladium (Ⅱ) Chloride (PdCl2) was purchased from Shaoyuan Co. Ltd. . Sodium borohydride (NaBH4) was obtained from Aladdin Bio-Chem Technology Co. Ltd. . All the above reagents were used directly without purification. Commercial honeycomb cordierite was bought from Huanya Chemical Packing Co. Ltd. . Before growth of catalysts, a commercial honeycomb cordierite (5 mm × 5 mm × 20 mm) was immersed and cleaned in the dilute nitric acid for ∼30 min, and rinsed with deionized water. Then it was dried at 80 °C overnight for later use.Sodium hydroxide (NaOH), ethylenediamine tetracetic acid disodium salt  was poured into a Teflon liner (25 ml), in which 1.5 mmol KMnO4 was dissolved to form a mixed solution under continuous stirring for ∼30 min. Then the clean cordierite substrate was put vertically on the bottom of liner. After hydrothermally maintained at 100°C for 12 h, the modified cordierite was carefully cleaned with deionized water and ethanol, then dried at 80°C for further use. The as-obtained monolithic sample was denoted as δ-MnO2-NFA.Uniform δ-MnOx wt% Pd/δ-MnO2 nanoflower arrays monolithic samples  were prepared using the electroless plating route . 2) Chemical plating of Pd. Then the activated δ-MnO2-NFA sample was put in the 50 ml platinum plating solution at 45–55°C for 4 h . The usage of PdCl2 was based on the theoretical Pd loading. 3) Drying. After being cleaned with deionized water, the resulting monolithic sample was dried at 80°C overnight. The x wt% Pd/δ-MnO2 nanoflower arrays monolithic sample was denoted as xPd/δ-MnO2-NFA. The loading mass of Pd/δ-MnO2 nanoflower arrays is ∼1 mg.A series of ng route . 1) Acti2 nanoflower arrays). The as-prepared 0.05 wt% Pd nanoparticles monolithic sample was denoted as 0.05Pd-NP.For comparison, the pure cordierite substrate supported Pd nanoparticles with 0.05 wt% theoretical loading amount was prepared by the electroless plating route under the same procedures described above  and transmission electron microscope . The average particle size of Pd NPs was obtained by counting at least 100 single Pd NPs from the FESEM image. Brunauer-Emmett-Teller (BET) surface areas of the monolithic samples were acquired using Quantachrome Autosorb iQ instrument at −196°C. Inductively coupled plasma mass spectroscopy (ICP-MS) technique was applied to acquire the actual Pd amount in the monolithic samples containing Pd-based species. Surface compositions and their binding energies of the samples were analyzed using X-ray photoelectron spectroscopy  equipped with Al Kα source.Structure identification of the monolithic samples was carried out by X-ray diffractometer  using Cu Ko-xylene combustion was performed in a quartz tube (Φ = 8 mm). Typically, the as-prepared monolithic samples with 16 channels (5 mm × 5 mm × 10 mm) were utilized to evaluate the catalytic performance. The gaseous reactant was consisted of 250 ppm toluene, O2 (20%) and N2 , and the total flow rate is 51 ml/min. Hence the GHSV is 10,000 h−1. Catalytic oxidation measurement was conducted in a temperature range of 110–300°C using a heating furnace. The concentrations of outlet gases were monitored in real time by a gas chromatograph (Agilent 6820) with flame ionization detector (FID), and no other products were found except CO2 and H2O. Therefore, the toluene or o-xylene conversion can be calculated by the consumption of toluene or o-xylene. The values of activation energies (Ea) were computed based on the equation (ln k = −Ea/RT + ln A) derived from the Arrhenius equation, where k is the rate constant (s−1) and A is the pre-exponential factor. The Ea values were calculated with toluene conversion below 15%.The catalytic activity test for toluene or 2-NFA samples was firstly investigated  are used to evaluate the catalytic performance of Pd/δ-MnO2-NFA samples  are nearly close to those of 0.07Pd/δ-MnO2-NFA. Considering both Pd2+ dosage and catalytic activity, Pd/δ-MnO2-NFA with 0.05 wt% Pd loading is the preferred sample for further investigation.The effect of Pd loading (from 0.01 to 0.07 wt%) on the catalytic activities for the Pd/δ-MnOstigated . For com samples . With th2-NFA, 0.05Pd-NP, δ-MnO2-NFA, and cordierite substrate. Obviously, the cordierite substrate exhibits no reactivity for toluene oxidation in the temperature range of 110–300°C. For the δ-MnO2-NFA sample, the toluene conversion is pretty low and only ∼20% toluene conversion is detected at 290°C. With only Pd loading, the 0.05Pd-NP sample exhibits a dramatically superior activity compared to the δ-MnO2-NFA sample, with T10, T50, and T90 at 226, 258, and 281°C, respectively. Depositing Pd NPs onto the δ-MnO2-NFA surface further improves the catalytic performance toward toluene combustion. The remarkable toluene oxidation activity in the 0.05Pd/δ-MnO2-NFA sample might be due to the strong synergy between Pd NPs and δ-MnO2-NFA support (T90) over the 0.05Pd/δ-MnO2-NFA sample is further compared with other Pd-based monolithic integrated catalysts reported in the previous studies  than the 0.20 wt% Pd-AlOOH/Al (T90 = 233°C) and 0.05 wt% Pd-AlOOH/Al-EDTA catalysts (T90 = 230°C)  . When thcatalyst .2-NFA, 0.05Pd-NP, and δ-MnO2-NFA. The Ea was computed by the linear-fitting plots (ln k vs. 1/T). As summarized in Ea values follow the order: δ-MnO2-NFA (54.1 kJ/mol) > 0.05Pd-NP (48.7 kJ/mol) > 0.05Pd/δ-MnO2-NFA (29.9 kJ/mol), showing the reverse order when comparing to the sequence of their catalytic performance. The lower Ea value means the easier toluene combustion  and 221°C (T90). Impressively, the toluene conversion for the 0.05Pd/δ-MnO2-NFA sample is not obviously changed  are observed for the 0.05Pd/δ-MnO2-NFA, δ-MnO2-NFA, and 0.05Pd-NP samples. Note that two distinct diffraction peaks at 12.4° and 24.8° appear in the 0.05Pd/δ-MnO2-NFA and δ-MnO2-NFA (2 (PDF No. 42-1317). Moreover, no diffraction peaks associated with Pd-based species such as Pd or PdO can be detected in the 0.05Pd/δ-MnO2-NFA and 0.05Pd-NP, which is likely due to the low loading of Pd-based components  is composed of many nanosheets with a thickness of ∼5 nm, offering a uniformly hierarchical and open structure for the deposition of Pd NPs. 2-NFA sample. Clearly, the Pd NPs with an average size of 11 ± 3 nm are highly dispersed over the surface of δ-MnO2 nanoflowers. However, when using cordierite substrate as the support, distinct change in size for the Pd NPs is observed in the 0.05Pd-NP sample , which is higher than that of pure cordierite substrate (3.21 m2/g). Additionally, the ICP-MS result confirms that the actual Pd loading amount of 0.05Pd/δ-MnO2-NFA (0.030 wt%) is more than that of 0.05Pd/NP (0.021 wt%). Compared with the pure cordierite substrate, the δ-MnO2-NFA sample with increased surface area is more beneficial for the dispersion of Pd NPs with smaller size, thus improving the utilization efficiency of Pd2+ precursor  = 642.3 and 643.4 eV, assignable to the surface Mn3+ and Mn4+ cations, respectively (3+/Mn4+ ratio rises from 1.33 (δ-MnO2-NFA) to 3.00 (0.05Pd/δ-MnO2-NFA), i.e., 0.05Pd/δ-MnO2-NFA owns more surface Mn3+ than the δ-MnO2-NFA   species, surface-absorbed oxygen (OSA) species, and hydroxyl oxygen (OOH) species, respectively (SA/OSL ratio decreases in the order of 0.05Pd/δ-MnO2-NFA (0.86) > 0.05Pd-NP (0.76) > δ-MnO2-NFA (0.42)  . From Fiectively . The OSAA (0.42) , which i2 nanoflowers existed in the 0.05Pd/δ-MnO2-NFA sample. Such a strong synergy is probably because of the electron transfer proceeding at the interface between Pd and δ-MnO2 (Pd0 + 2Mn4+ → Pd2+ + 2Mn3+)  , leading species . As is wformance . Therefo2 and toluene, thereby enhancing the reactivity in toluene combustion  on the Pd-supported catalysts is based on the Mars-van Krevelen mechanism (2-NFA sample is depicted in via the Pdx+ species (Pd2+ and Pd0). (Step 2) The adsorbed toluene is activated and oxidized by OSL from the 0.05Pd/δ-MnO2-NFA surface, which then completely converts into CO2 and H2O. Meanwhile, the surface of 0.05Pd/δ-MnO2-NFA is reduced, thus lowing the valence of surface Mn4+ and Pd2+ ions and generating the surface oxygen vacancies. (Step 3) The reduced surface of 0.05Pd/δ-MnO2-NFA is timely re-oxidized by OSA supplied from gaseous O2.It has been reported that benzene series  and o-xylene  combustion at a GHSV of 10,000 h−1, respectively. Furthermore, the 0.05Pd/δ-MnO2-NFA sample demonstrated the stable activity in a 30 h long-term test. Our work affords useful guidance for rational design of monolithic integrated catalysts toward high-efficiency VOCs elimination.In this work, honeycomb cordierite coated with uniform δ-MnO


Saccharomyces cerevisiae, and is widely used in the pharmaceutical, food, and cosmetic industries. It has been reported that addition of l-serine (l-Ser) is effective at increasing the intracellular glutathione content because l-Ser is the common precursor of l-cysteine (l-Cys) and glycine (Gly) which are substrates for glutathione biosynthesis. Therefore, we tried to enhance the l-Ser biosynthetic pathway in S. cerevisiae for improved glutathione production.Glutathione is a valuable tri-peptide that is industrially produced by fermentation using the yeast SER2, SER1, SER3, and SER33 involved in l-Ser biosynthesis at 48 h cultivation was increased 1.3, 1.4, 1.9, and 1.9-fold, respectively, compared with that of the host GCI strain, which overexpresses genes involved in glutathione biosynthesis. We further examined simultaneous overexpression of SHM2 and/or CYS4 genes involved in Gly and l-Cys biosynthesis, respectively, using recombinant GCI strain overexpressing SER3 and SER33 as hosts. As a result, GCI overexpressing SER3, SHM2, and CYS4 showed the highest volumetric glutathione production (64.0 ± 4.9 mg/L) at 48 h cultivation, and this value is about 2.5-fold higher than that of the control strain.The volumetric glutathione production of recombinant strains individually overexpressing l-Ser and Gly biosynthetic pathway are useful strategies for fermentative glutathione production by S. cerevisiase.This study first revealed that engineering of The online version contains supplementary material available at 10.1186/s12934-022-01880-8. Glutathione is the most abundant non-protein thiol compound in all living organisms . BecauseGSH1, and glutathione synthetase , encoded by GSH2, from three precursor amino acids, l-glutamate (l-Glu), l-cysteine (l-Cys), and glycine (Gly)  that showed increased glutathione production , which was previously constructed from a YPH499 strain by expressing multiple copies of GSH1 and GSH2 genes by the δ-integration method [E. coli strains were grown in LB medium  supplemented with ampicillin (100 mg/L). Recombinant S. cerevisiae GCI strains were grown in SD  medium. Adenine (20 mg/L), histidine (20 mg/L), tryptophan (40 mg/L), leucine (100 mg/L), uracil (20 mg/L) and aureobasidin A (0.5 mg/L) were supplemented as necessary.n method , was usel-Ser biosynthetic pathway  were amplified by polymerase chain reaction (PCR) method from S. cerevisiae chromosomal deoxyribonucleic acid (DNA) using primer sets of SER2F1/R1, SER1F1/R1, SER3F1/R1, and SER33F1/R1, respectively  and l-Cys biosynthetic pathway (CYS4) were amplified by PCR method from S. cerevisiae chromosomal DNA using primer sets of SHM2F/R, and CYS4F/R, respectively  for 24 h. An adequate volume of each cell culture was inoculated into 20 mL of the same medium in 200 mL of baffled erlenmeyer flask with or without 300 mg/L l-Ser to achieve an initial OD600 value of 0.15. Cells were then grown at 30 °C with agitation (200 rpm) for up to 72 h.Glycerol stocks of recombinant S. cerevisiae cells were determined as described in our previous report [600 of the culture samples were measured using a UVmini-1240 Spectrometer . The cells were pelleted by centrifugation  and rinsed with Milli-Q water twice. The rinsed cell pellets were re-suspended in Milli-Q water and incubated at 95 °C for 3 min, then cooled immediately on ice for 3 min and the supernatant was separated by centrifugation . GSH and GSSG concentrations in the supernatant were determined by HPLC (Shimadzu) equipped with a YMC-Pack ODS-A column . The operating conditions were 30 °C with 50 mM potassium dihydrogen phosphate buffer (pH 2.8) and 10 mM sodium 1-heptanesulfonate as the mobile phase at a flow rate of 1.0 mL/min, and detection was performed with an ultraviolet detector SPD-20A (Shimadzu) at 210 nm. Volumetric glutathione production (mg/L-broth) and intracellular glutathione content (%) were calculated from OD600 and used to determine glutathione concentration, as previously described [Reduced glutathione (GSH) and oxidized glutathione (GSSG) in recombinant s report . Cell cuAdditional file 1: Table S1. Primers used in this study. Table S2. Data used in Fig.  Table S3 Data used in Fig.  Table S4 Data used in Fig.  Table S5 Data used in Fig.


Tristaniopsis laurina which led to the discovery of a new anti-inflammatory compound, tristaenone A (1). The structure was elucidated by detailed spectroscopic data analysis, and the absolute configuration was established using X-ray crystallography analysis. Tristaenone A (1) suppressed LPS and IFN-γ-induced NO, TNF-α and IL-6 production in RAW 264.7 cells with IC50 values of 37.58 ± 2.45 μM, 80.6 ± 5.82 μM and 125.65 ± 0.34 μM, respectively. It also inhibited NF-κB nuclear translocation by 52.93 ± 14.14% at a concentration of 31.85 μM.Inspired by ethnopharmacological knowledge, we conducted a bioassay-guided fractionation of the leaves of  Inflammation is a biological response to various stimuli such as pathogens, damaged cells, toxic compounds, pollutants or irradiation . ProlongMacrophages are essential immune cells that play a crucial role in the process of inflammation. Macrophages on activation produce large amounts of various pro-inflammatory mediators, such as nitric oxide (NO), prostaglandin-E2 (PGE2), pro-inflammatory cytokine ) and tumor necrosis factor-alpha (TNF-α) . StudiesTraditional remedies and natural products are an alternative to clinical drugs which have severe side effects, and they hold considerable promise in terms of identifying bioactive lead molecules and developing them into therapeutics to treat inflammatory illnesses ,8. AustrTristaniopsis laurina (Myrtaceae) (Peter G. Wilson and J.T Waterh) based on existing ethnopharmacological knowledge documented by Francis Bodkin on its use to heal sores and ulcers 25D −179.8 ; UV-Vis λmax (MeOH) nm (log ε) 280 (5.34) and 233 (5.26); 1D and 2D NMR  data .data see ; HRESI (tristaenone A (CCDC 2172244) was deposited at the Cambridge Crystallographic Data Center. Additional crystallographic information is available in the Single crystal data was collected on the MX1 beamline at the Australian Synchrotron, using silicon double crystal monochromatic radiation (λ = 0.71073 Å) at 100 K . The XDS1: C24H24O5 (M = 392.43 g/mol); monoclinic, 0.1 × 0.1 × 0.01 mm3, space group P21,V = 991.0(4) Å3, Z = 2, Dc = 1.315 g/cm3, F(000) = 416.0, Mo Kα radiation, λ = 0.71073 Å, T = 100 K, μ = 0.092 mm–1; 2θrange = 50.5°, 12,424 reflections collected, 3855 unique (Rint = 0.0453); final GooF = 1.066, R1 = 0.0323 [I > 2σ(I)], wR2 = 0.0825; absolute structure parameter = −0.1(3). Crystal data for 2 flasks on DMEM containing 10% fetal bovine serum (FBS) that was supplemented with penicillin (100 μ/mL), streptomycin (100 μg/mL) and L-glutamine (2 mM). The cell line was maintained in 5% CO2 at 37 °C, with media being replaced every 3–4 days. Once cells had grown to confluence in the culture flask, they were removed using a rubber policeman, as opposed to using trypsin, which can remove membrane-bound receptors.Cells were grown in 75 cm6 cells/mL) were seeded in 96 well plates  overnight until confluency. When the cells were confluent, each compound was serially diluted from 100 μg/mL in two-fold dilution steps and co-incubated with cells for 1 h prior to the addition of 1 μg/mL LPS and 10 U/mL (1 unit = 0.1 ng/mL) IFN-γ. After activation, the cells were incubated for another 24 h at 37 °C. The supernatant was then collected for NO, TNF-α and IL-6 assays. The cells were subjected to cell viability measurements using the Alamar Blue assay. Non-activated cells (exposed to media only) were used as negative control and activated cells were positive control.RAW 264.7 cells  according to the manufacturer’s instructions. The absorbance was measured at 410 nm . The con1 at 31.85, 63.70 and 127.41 μM in serum-free medium DMEM for 1 h prior to the stimulation of a combination of 1 μg/mL LPS and 10 U/mL (1 unit = 0.1 ng/mL) IFN-γ for 30 min. Cells were then washed with pre-chilled phosphate-buffered saline (PBS) buffer  and fixed with 4% paraformaldehyde  for 30 min at room temperature. Triton X 100  was added to permeabilize the cells for 20 min before being washed again three times with PBS and then blocked with 3% of bovine serum albumin  for 1 h. The mouse anti-p65 NF-κB antibody  was co-incubated with cells overnight at 4 °C. Cells were rinsed (three times) with PBS again and incubated with the donkey anti-mouse IgG conjugated with Alexa Fluor 488  for 1 h in the dark room at room temperature. After washing with PBS, the chambers were removed and the anti-fade mounting media with DAPI solution  was added before capturing images with the Inverted Leica TCS SP5 laser scanning confocal microscope . The fluorescent intensity was quantified and analysed using ImageJ following the previous study  was added to cells and incubated at 37 °C for 2 h. The fluorescence intensity was measured with excitation at 530 nm and emission at 590 nm using a microplate reader. The results were expressed as a percentage of the intensity of that of control cells (non-activated cells).50 values were obtained by using the sigmoidal dose–response function in GraphPad Prism. The results were expressed as mean ± standard deviation (SD).Data analysis was carried out using GraphPad Prism 9.3.1. Calculations were performed using MS-Excel version 16.61.1. IC


The cations in the title salt show π-stacking. The chloride anions, together with the water molecules, form hydrogen-bonded zigzag chains. 10H8NS+·Cl−·H2O, obtained by the reaction of sodium quinoline-8-thiolate Na(Quin-8-S) with CH2Cl2 and an aqueous solution of [Bu4N]Cl, contains π-stacked cations [plane-to-plane separation = 3.338 (4)–3.356 (4) Å] and features chains built by alternating Cl− anions and H2O molecules connected by O—H⋯O hydrogen bonds. The cation shows whole-molecule disorder over two flipped orientations in a 0.853 (3):0.147 (3) ratio.The structure of the title hydrated molecular salt, C The solution was then extracted with CH2Cl2 giving a yellow organic phase, which was then evaporated yielding a yellow oil. After a few hours, yellow–orange crystals of 2H-thiazoloquinolin-3-ium chloride monohydrate had formed in the oil.During an attempt to obtain [BuCrystal data, data collection and structure refinement details are summarized in Table 210.1107/S2414314620014650/hb4368sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2414314620014650/hb4368Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2414314620014650/hb4368Isup3.molSupporting information file. DOI: 2042568CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Lower extremity infections in diabetic patients are associated with high rates of hospitalization and amputation. Given the microvascular dysfunction and poor peripheral circulation in this population, the aim of this study was to assess tebipenem soft tissue pharmacokinetics (PK) and interstitial fluid distribution among diabetic patients with lower extremity infections using This was a single-center, open-label, observational PK study in diabetic patients with foot infections who were enrolled and received tebipenem (600 mg) orally q8 hrs for a total of 3 doses. A microdialysis catheter was inserted within 4-8 cm of the wound margin to allow for dialysate sampling. Ten concurrent plasma and dialysate samples over an 8 hr period starting immediately prior to the last dose of tebipenem were obtained. Protein binding was determined by ultracentrifugation at 1 hr post-third dose. Plasma and dialysate samples were assayed via a validated LC/MS/MS assay. Non-compartmental analyses for free plasma and soft-tissue concentration were used to obtain PK parameters.fCmax), 2.34 ± 0.94 mg/L; time to Cmax (Tmax), 2.99 ± 1.40 hr; half-life (t1/2), 2.23 ± 1.65 hr and free area under the concentration-time curve (fAUCp(0-8)): 8.37 ± 1.63 mg.h/L. Mean ± SD parameters in tissue were: Cmax, 2.50 ± 1.04 mg/L; Tmax, 2.99 ± 1.40 h; t1/2 1.99 ± 1.42 hr; and AUCt(0-8), 8.01 ± 0.76 mg.h/L.Four male diabetic patients with an age of 70 ± 7 years and a hemoglobin A1C of 10 ± 2% were consented. All patients had an active complicated skin and soft tissue infection as defined by PEDIS Grade 2 or 3. Mean ± standard deviation (SD) plasma protein binding was 50% ± 1%. Mean ± SD tebipenem PK parameters in plasma were: maximum free concentration (This study demonstrates that tebipenem has excellent distribution into interstitial fluid and lower extremity tissue of diabetic patients with ongoing foot infections.Yasmeen Abouelhassan, PhD, Pfizer: spouse salary David P. Nicolau, PharmD, Shionogi: Grant/Research Support.


The formation of complexes with Gd(III) and Dy(III) ions involving fumarate and maleate groups of the polyester was proved by IR and electron spectroscopy methods. It was established that the structure of the coordination units has the form of a square antiprism. The compositions and conditional logarithms of the stability constants of the complexes were determined. It was established that complexation with lanthanide ions promotes emission enhancement in the ligand.For the first time, metal–polymer complexes have been synthesized using hyperbranched polyester polyfumaratomaleate as a matrix, the structure of which has been established by  Compounds of rare-earth elements are effectively used in contrasting reagents for MRI imaging of organ and tissue pathologies ,2,3. To The use of hyperbranched polymers with a 3D architecture and a high density of peripheral functional groups as a platform could be very promising for the creation and stabilization of gadolinium and dysprosium complexes. Hyperbranched polymers are densely packed macromolecules whose three-dimensional framework consists of a core and branching short chains—dendrons. One representative of such structures is hyperbranched polyesters of the Boltorn H series. These polymers consist of ethoxylated pentaerythritol as a core and elementary units formed by polycondensation of 2,2-bis(hydroxymethyl)propionic acid. These nanosized polyesters are industrially available, biodegradable, well-soluble in various polar solvents, and have low toxicity, and the reactive terminal hydroxyl groups can be easily modified to give them the desired set of properties. The introduction of coordinatively active functional groups made it possible to obtain polydentate ligands that form stable complexes with transition metal ions Co(II), Ni(II), and Cu(II) . It has Since the luminescence mechanism of lanthanide coordination compounds consists of light absorption by a ligand and energy transfer through its triplet level to the lanthanide ion, which emits it as an electromagnetic wave, the molecular design of luminophores is reduced to the search for suitable ligand–lanthanide pairs that form stable coordination compounds and have an optimal ligand triplet level for the excitation of the corresponding lanthanide ion resonance level . Due to The thermodynamic stability of complexes of lanthanides with derivatives of maleic and fumaric acids has been shown by numerous studies ,24,25,26−1, Perstorp Speciality Chemicals AB, Sweden), maleic anhydride , SnCl2 , Gd(NO3)3∙5H2O , and Dy(NO3)3∙5H2O . The organic solvents used were 1.4-dioxane, benzene, acetone, ethanol, methanol, and distilled water.We used second-generation Boltorn H hyperbranched polyester polyol with 16 terminal hydroxyl groups  in the wavelength range of 230~860 nm at T = 25 ± 0.01 °C, with an absorbing layer thickness of l = 1 cm. Elemental analysis was performed using a Euro EA 3000 CHNS analyzer . Luminescence spectra were recorded using a fluorescent spectrometer LS 55  with an excitation wavelength of 200 to 800 nm and an emission of 200 to 900 nm, excitation and emission slit of 5 nm, speed of 100 nm min−1 with weak gain at T = 25 ± 0.01 °C, and the thickness of the absorbing layer l = 1.5 cm. 3)3-compound 2: c2 = 4.1 × 10−4 − 5.1 × 10−5 mol L−1, cGd(NO3)3 = 5.1 × 10−4 − 1.0 × 10−4 mol L−1, λ = 255 nm, εcomplex = 7648.78 L∙cm−1∙mol−1, λ = 281 nm, εcomplex = 2715.26 L∙cm−1∙mol−1, λ = 304 nm, εcomplex = 1132.75 L∙cm−1∙mol−1; for the system Dy(NO3)3-compound 2: c2 = 4.1 × 10−4 − 5.1 × 10−5 mol L−1, cDy(NO3)3 = 5.1 × 10−4 − 1.0 × 10−4 mol L−1, λ = 262 nm, εcomplex = 6423.92 L∙cm−1∙mol−1, λ = 279 nm, εcomplex = 2997.41 L∙cm−1∙mol−1, λ = 302 nm, εcomplex = 1202.28 L∙cm−1∙mol−1.The composition and stability of complexes in the lanthanide salt–sodium salt systems of hyperbranched polyester polyfumaratomaleate were determined by electron absorption spectra using Job’s Method ,28. The 2 (0.132 mmol) as a catalyst in 4 mL of 1.4-dioxane was added to the solution. The reaction was carried out under stirring and at 100 °C for 36 h. The product was precipitated with benzene and dried in a thermal vacuum cabinet at 50 °C and a vacuum of 1 mmHg until constant weight. Compound 1 was a light-yellow resin with a yield of 57%.A 2.50 g (1.43 mmol) sample of second-generation hyperbranched polyester polyol was incubated at 140 °C for 40 min for dehydration and destruction of the self-associates. After cooling to 50 °C, the polymer was dissolved in 10 mL of 1.4-dioxane, then a solution of 1.96 g (19.99 mmol) maleic anhydride and 0.025 g SnCI1H NMR spectrum [(CD3)2SO], δ ppm: 0.90–1.27 ; 3.29–3.66 ; 3.95–4.36 ); 6.22–6.32 ; 6.37–6.46 . IR spectrum, ν, cm−1: 3712−3118 (O−Ha.); 3712−3118, 2777, 2587 (O−Hc.a.); 3095, 3064 (C−HC=C); 2970, 2923, 2890, 2863 ; 1735 ; 1640 (C=Ccis); 1467, 1417, 1377 [δ ]; 1417, 685, 644, 614, 412 [δ ]; 1296−1009 ; 918 [δ (O−Hc.a)]; 894, 868, 820 [δ ]. Elemental analysis. Found, %: C 50.42; H 5.18. C121H148O80. Calculated, %: C 51.11; H 4.89. UV–Vis spectrum (DMSO), λmax, nm : 260 ROH n→σ*, CH=CH π→π*, RC(O)OR’ n→π* [3601]; 288 C(O)OH n→π* [614.3].1 was dissolved in 10 mL acetone and 0.019 molar NaHCO3 in 5 mL ethanol/water (1:1) was added. Product 2 was separated by filtration and dried in a thermal vacuum cabinet at 50 °C and a vacuum of 1 mmHg until constant weight. Compound 2 was a light-yellow powder with a yield of 65%.A sample of 1.50 g (0.513 mmol) of compound −1: 3685−3078 (O−Ha.); 3101 (C−HC=C); 2955−2855 ; 1732 (C=Oester); 1593, 1423 (C(O)O−); 1469−1358 [δ ]; 1423, 671, 609, 420 [δ ]; 1304−1014 ; 879, 833, 817 [δ ]. Elemental analysis. Found, %: C 46.19; H 4.36; Na 8.77. C121H136Na12O80. Calculated, %: C 46.55; H 4.05; Na 8.34. UV–Vis spectrum (DMSO), λmax, nm : 272 ROH n→σ*, CH=CH π→π*, RC(O)OR’ n→π* [3866]; 305 C(O)O− n→π* [1009].IR spectrum, ν, cm3)3∙5H2O and Dy(NO3)3∙5H2O in 5 mL of methanol were added to solutions of 0.5 g (0.157 mmol) of product 2 in 2 mL of methanol 0.0022. The isolated complexes were dried in a thermal vacuum oven at 50 °C and a vacuum of 1 mmHg until constant weight. Molar solutions of Gd; 3063 (C−HC=C); 2959−2854 ; 1732 (C=Oester.); 1647, 774 [δ (NO3−)]; 1577, 1508−1342 (C(O)O−); 1508−1342 [δ ]; 1508−1342, 671, 609, 420 [δ ]; 1300−1006 ; 856, 814 [δ ]. Elemental analysis. Found, %: C 38.14; H 4.06; S, 6.46; Gd 15.85. C126H160Gd4O88S8. Calculated, %: C 37.87; H 4.11; S, 6.13; Gd 16.03. UV–Vis spectrum (DMSO), λmax, nm: 254 ROH n→σ*, CH=CH π→π*, RC(O)OR’ n→π*; 304 C(O)O− n→π*, Gd(III) 8S7/2→6P7/2, 6P5/2, 6I7/2, 6I9/2, 6I17/2, 6I11/2, 6I13/2, 6I15/2, 6D9/2. The Gd(III) complex 4 is an amorphous compound of light-beige color, 89% yield. IR spectrum, ν, cm−1: 3692−3036 (O−Ha.); 3105 (C−HC=C); 2959−2858 ; 1732 (C=Oester.); 1643, 814, 714 [δ (NO3−)]; 1585, 1462−1350 (C(O)O−); 1462−1350 [δ ]; 1462−1350, 663, 609, 428 [δ ]; 1304−1006 ; 852, 814 [δ ]. Elemental analysis. Found, %: C 36.43; H 3.89; S, 11.31; Dy 15.28. C129H164Gd4O88S15. Calculated, %: C 36.09; H 4.11; S, 11.43; Dy 15.36. UV–Vis spectrum (DMSO), λmax, nm: 260 ROH n→σ*, CH=CH π→π*, RC(O)OR’ n→π*; 302 C(O)O− n→π*; 350 Dy(III) 6H15/2→6P7/2; 365 Dy(III) 6H15/2→4P5/2; 387 Dy(III) H15/2→4I13/2; 450 Dy(III) 6H15/2→4I15/2; 758 Dy(III) 6H15/2→6F3/2; 808 Dy(III) 6H15/2→6F5/2.The Dy(III) complex 1 was synthesized by the reaction of second-generation hyperbranched polyester polyol with maleic anhydride  4 were obtained  into fumarate groups (6.46–6.37 ppm) in the macromolecule of compound tely 50% .1 contains six fumarate and six maleate fragments.The degree of functionalization of end hydroxyl groups of hyperbranched polyester polyol was estimated by calculating the ratio of the integral signal intensities of olefin and methyl groups of the polyester complex ester skeleton. It was found that the degree of functionalization is 75%; compound 1  and 685, 644, 614 cm−1 (cis-isomer). An approximation of the absorption bands of compound 1 at 1735 and 1640 cm−1 by the Gaussian method showed the presence of bands of valent vibrations of C=O bonds in the ester and acidic fragments at 1738 and 1695 cm−1, and the bands at 1648 and 1632 cm−1 correspond to valent vibrations of C=C bonds in the fumarate and maleate groups O– and C=O bonds at 1577, 1508–1342 and 1041, 1006 cm−1. In addition, when comparing the IR spectra of the Dy(III) complex (3) with compound (2), an increase in the intensity of the bands of the stretching vibrations of the C=O bond in the ester group at 1732 cm−1 and a decrease in the intensity of the bands of the stretching vibrations of the C(O)O– and C=O bonds at 1585, 1462–1350 and 1304–1006 cm−1.A comparison of the IR spectra of the Gd(III) complex (3) with compound (2)  demonstr3 and 4 are in coordination with the oxygen atoms of the carboxylate anionic and ester groups. In addition, the coordination sites of complexes 3 and 4 contain nitro groups at 1647, 774, and 1643, 814, 714 cm−1.All these facts suggest that the Gd(III) and Dy(III) ions in complexes 1 and its complexes with Gd(III) and Dy(III) ions were studied by spectrophotometry. The obtained plots were subjected to interpolation by the Gaussian distribution function, and the results of the analysis are presented in Hyperbranched polyester polyfumaratomaleate 1 solution in DMSO  ion from the 8S7/2 level to the 6P7/2, 6P5/2,6 I7/2, 6I9/2 6I17/2, 6I11/2, 6I13/2, 6I15/2, 6D9/2 levels, indicating the presence of eight coordinated Gd(III) ions in the DMSO solution  from 6H15/2 to 6P7/2, 4P5/2, 4I13/2, 4I15/2, 6F3/2 and 6F5/2, respectively . T. T1 and ent used B. The peow–green ,53. The 1 in a 57% yield. The structure of compound 1 was proved by 1H NMR, IR and electron spectroscopy, and the presence of six fumarate and six maleate fragments at terminal positions was established. The hyperbranched terminally decorated polyester (1) was successfully used as a polydentate ligand for doping with Gd(III) and Dy(III) rare-earth cations.The reaction of the addition of maleic anhydride to hyperbranched second-generation polyester polyol obtained hyperbranched polyester polyfumaratomaleate 3 and Dy(III) 4 ions were synthesized based on ligand 1. The oxygen atoms of fumarate and maleate groups participate in complexation with Gd(III) and Dy(III) ions by IR and electron spectroscopy. According to UV–Vis spectrophotometry, three carboxyl groups of the terminal fumarate or maleate fragments are involved in the coordination of one metal ion. The Gd(III) and Dy(III) coordination sites have the composition [MO6X2] (M= Gd(III), Dy(III), X= NO3− or H2Ocoordination) and have a quadratic-antiprismatic geometry. The logarithms of the stability constants are lgβ = 4.59 ± 0.26 and 4.84 ± 0.07, respectively. The complexation with Gd(III) and Dy(III) ions promotes the luminescence enhancement in comparison with ligand 1 by 16.77 and 202.08 times. The significant difference in the emission intensity of the complex with Dy(III) ions 4 as compared with complex with Gd(III) ions 3 (approximately 18 times) appears to be due to the fact that, in Gd(III), in the first excited state 6P7/2, Gd(III) is too far away to accept the energy on the triplet excited state of the ligand [New complexes with Gd(III) e ligand ,53. The