cometadata/datacite-titles-descriptions-related-identifiers huggingface dataset:
DOI: 10.6084/m9.figshare.1568567.v2
title: Effect of organic cultivation of rooibos tea plants (<i>Aspalathus linearis</i>) on soil nutrient status in Nieuwoudtville, South Africa

description: The shoots of rooibos (<i>Aspalathus linearis</i> (Burm.f.) R.Dahlgren) plants, cultivated organically by small-scale farmers in Nieuwoudtville, are harvested for the production of tea. These practices could lead to decreasing soil fertility. It was hypothesised that soil from cultivated rooibos plots will have lower nutrient concentrations than soil from adjacent uncultivated plots. Soil and shoot samples were collected in December 2005, 2006 and 2009 from cultivated fields of increasing plot age and from adjacent uncultivated plots on three farms, and analysed for nutrient concentration. Compared with the uncultivated plots, no measured soil nutrients including concentrations of phosphorus (P), exchangeable potassium (K), magnesium (Mg) and calcium (Ca), and soil carbon (C) decreased in cultivated plots over the five-year period of assessment. Soil C correlated positively with concentrations of soil exchangeable K, Mg and Ca, and sodium (<i>P</i>&lt;0.001), indicating that soil C is an important indicator of soil fertility. Foliar P increased, and consequently the nitrogen:P ratio decreased in cultivated relative to uncultivated plants, implying higher P-uptake by cultivated plants. Overall, organic rooibos cultivation in Nieuwoudtville appears to be sustainable in terms of maintaining soil nutrition because soil nutrient status did not decrease over the five-year period.
DOI: 10.6084/m9.figshare.1568561
title: Progesterone 5β-reductases/iridoid synthases (PRISE): gatekeeper role of highly conserved phenylalanines in substrate preference and trapping is supported by molecular dynamics simulations

description: Vein Patterning 1 (<i>VEP1</i>)-encoded progesterone 5β-reductases/iridoid synthases (PRISE) belong to the short-chain dehydrogenase/reductase superfamily of proteins. They are characterized by a set of highly conserved amino acids in the substrate-binding pocket. All PRISEs are capable of reducing the activated C=C double bond of various enones enantioselectively and therefore have a potential as biocatalysts in bioorganic synthesis. Here, recombinant forms of PRISEs of <i>Arabidopsis thaliana</i> and <i>Digitalis lanata</i> were modified using site-directed mutagenesis (SDM). In r<i>Dl</i>P5βR, a set of highly conserved amino acids in the vicinity of the catalytic center was individually substituted for alanine resulting in considerable to complete loss of enone reductase activity. F153 and F343, which can be found in most PRISEs known, are located at the outer rim of the catalytic cavity and seem to be involved in substrate binding and their role was addressed in a series of SDM experiments. The wild-type PRISE accepted progesterone (large hydrophobic 1,4-enone) as well as 2-cyclohexen-1-one (small hydrophilic 1,4-enone), whereas the double mutant r<i>At</i>P5βR_F153A_F343A converted progesterone much better than the wild-type enzyme but almost lost its capability of reducing 2-cyclohexen-1-one. Recombinant <i>Draba aizoides</i> P5βR (r<i>Da</i>P5βR) has a second pair of phenylalanines at position 156 and 345 at the rim of the binding site. These two phenylalanines were introduced into r<i>At</i>P5βR_F153A_F343A and the resulting quadruple mutant r<i>At</i>P5βR_F153A_F343A_V156F_V345F partly recovered the ability to reduce 2-cyclohexen-1-one. These results can best be explained by assuming a trapping mechanism in which phenylalanines at the rim of the substrate-binding pocket are involved. The dynamic behavior of individual P5βRs and mutants thereof was investigated by molecular dynamics simulations and all calculations supported the ‘gatekeeper’ role of phenylalanines at the periphery of the substrate-binding pocket. Our findings provide structural and mechanistic explanations for the different substrate preferences seen among the natural PRISEs and help to explain the large differences in catalytic efficiency found for different types of 1,4-enones.
DOI: 10.6084/m9.figshare.1568565.v1
title: Pharmacological inhibition of lysosomes activates the MTORC1 signaling pathway in chondrocytes in an autophagy-independent manner

description: Mechanistic target of rapamycin (serine/threonine kinase) complex 1 (MTORC1) is a protein-signaling complex at the fulcrum of anabolic and catabolic processes, which acts depending on wide-ranging environmental cues. It is generally accepted that lysosomes facilitate MTORC1 activation by generating an internal pool of amino acids. Amino acids activate MTORC1 by stimulating its translocation to the lysosomal membrane where it forms a super-complex involving the lysosomal-membrane-bound vacuolar-type H<sup>+</sup>-ATPase (v-ATPase) proton pump. This translocation and MTORC1 activation require functional lysosomes. Here we found that, in contrast to this well-accepted concept, in epiphyseal chondrocytes inhibition of lysosomal activity by v-ATPase inhibitors bafilomycin A<sub>1</sub> or concanamycin A potently activated MTORC1 signaling. The activity of MTORC1 was visualized by phosphorylated forms of RPS6 (ribosomal protein S6) and EIF4EBP1, 2 well-known downstream targets of MTORC1. Maximal RPS6 phosphorylation was observed at 48-h treatment and reached as high as a 12-fold increase (p&lt;0.018). This activation of MTORC1 was further confirmed in bone organ culture and promoted potent stimulation of longitudinal growth (p&lt;0.001). Importantly, the same effect was observed in ATG5 (autophagy related 5)-deficient bones suggesting a macroautophagy-independent mechanism of MTORC1 inhibition by lysosomes. Thus, our data show that in epiphyseal chondrocytes lysosomes inhibit MTORC1 in a macroautophagy-independent manner and this inhibition likely depends on v-ATPase activity.
DOI: 10.6084/m9.figshare.1568561.v2
title: Progesterone 5β-reductases/iridoid synthases (PRISE): gatekeeper role of highly conserved phenylalanines in substrate preference and trapping is supported by molecular dynamics simulations

description: Vein Patterning 1 (<i>VEP1</i>)-encoded progesterone 5β-reductases/iridoid synthases (PRISE) belong to the short-chain dehydrogenase/reductase superfamily of proteins. They are characterized by a set of highly conserved amino acids in the substrate-binding pocket. All PRISEs are capable of reducing the activated C=C double bond of various enones enantioselectively and therefore have a potential as biocatalysts in bioorganic synthesis. Here, recombinant forms of PRISEs of <i>Arabidopsis thaliana</i> and <i>Digitalis lanata</i> were modified using site-directed mutagenesis (SDM). In r<i>Dl</i>P5βR, a set of highly conserved amino acids in the vicinity of the catalytic center was individually substituted for alanine resulting in considerable to complete loss of enone reductase activity. F153 and F343, which can be found in most PRISEs known, are located at the outer rim of the catalytic cavity and seem to be involved in substrate binding and their role was addressed in a series of SDM experiments. The wild-type PRISE accepted progesterone (large hydrophobic 1,4-enone) as well as 2-cyclohexen-1-one (small hydrophilic 1,4-enone), whereas the double mutant r<i>At</i>P5βR_F153A_F343A converted progesterone much better than the wild-type enzyme but almost lost its capability of reducing 2-cyclohexen-1-one. Recombinant <i>Draba aizoides</i> P5βR (r<i>Da</i>P5βR) has a second pair of phenylalanines at position 156 and 345 at the rim of the binding site. These two phenylalanines were introduced into r<i>At</i>P5βR_F153A_F343A and the resulting quadruple mutant r<i>At</i>P5βR_F153A_F343A_V156F_V345F partly recovered the ability to reduce 2-cyclohexen-1-one. These results can best be explained by assuming a trapping mechanism in which phenylalanines at the rim of the substrate-binding pocket are involved. The dynamic behavior of individual P5βRs and mutants thereof was investigated by molecular dynamics simulations and all calculations supported the ‘gatekeeper’ role of phenylalanines at the periphery of the substrate-binding pocket. Our findings provide structural and mechanistic explanations for the different substrate preferences seen among the natural PRISEs and help to explain the large differences in catalytic efficiency found for different types of 1,4-enones.
DOI: 10.6084/m9.figshare.1568565.v2
title: Pharmacological inhibition of lysosomes activates the MTORC1 signaling pathway in chondrocytes in an autophagy-independent manner

description: Mechanistic target of rapamycin (serine/threonine kinase) complex 1 (MTORC1) is a protein-signaling complex at the fulcrum of anabolic and catabolic processes, which acts depending on wide-ranging environmental cues. It is generally accepted that lysosomes facilitate MTORC1 activation by generating an internal pool of amino acids. Amino acids activate MTORC1 by stimulating its translocation to the lysosomal membrane where it forms a super-complex involving the lysosomal-membrane-bound vacuolar-type H<sup>+</sup>-ATPase (v-ATPase) proton pump. This translocation and MTORC1 activation require functional lysosomes. Here we found that, in contrast to this well-accepted concept, in epiphyseal chondrocytes inhibition of lysosomal activity by v-ATPase inhibitors bafilomycin A<sub>1</sub> or concanamycin A potently activated MTORC1 signaling. The activity of MTORC1 was visualized by phosphorylated forms of RPS6 (ribosomal protein S6) and EIF4EBP1, 2 well-known downstream targets of MTORC1. Maximal RPS6 phosphorylation was observed at 48-h treatment and reached as high as a 12-fold increase (p &lt; 0.018). This activation of MTORC1 was further confirmed in bone organ culture and promoted potent stimulation of longitudinal growth (p &lt; 0.001). Importantly, the same effect was observed in ATG5 (autophagy-related 5)-deficient bones suggesting a macroautophagy-independent mechanism of MTORC1 inhibition by lysosomes. Thus, our data show that in epiphyseal chondrocytes lysosomes inhibit MTORC1 in a macroautophagy-independent manner and this inhibition likely depends on v-ATPase activity.
DOI: 10.6084/m9.figshare.1568561.v1
title: Progesterone 5β-reductases/iridoid synthases (PRISE): gatekeeper role of highly conserved phenylalanines in substrate preference and trapping is supported by molecular dynamics simulations

description: Vein Patterning 1 (<i>VEP1</i>)-encoded progesterone 5β-reductases/iridoid synthases (PRISE) belong to the short-chain dehydrogenase/reductase (SDR) super family of proteins. They are characterized by a set of highly conserved amino acids in the substrate binding pocket. All PRISEs are capable of reducing the activated C=C double-bond of various enones enantioselectively and therefore have a potential as biocatalysts in bioorganic synthesis. Here, recombinant forms of PRISEs of <i>Arabidopsis thaliana</i> and <i>Digitalis lanata</i> were modified using site-directed mutagenesis (SDM). In r<i>Dl</i>P5βR a set of highly conserved amino acids in the vicinity of the catalytic center were individually substituted for alanine resulting in considerable to complete loss of enone reductase activitiy. F153 and F343, which can be found in most PRISEs known, are located at the outer rim of the catalytic cavity and seem to be involved in substrate binding and their role was addressed in a series of SDM experiments. The wild-type PRISE accepted progesterone (large hydrophobic 1,4-enone) as well as 2-cyclohexen-1-one (small hydrophilic 1,4-enone) whereas the double mutant r<i>At</i>P5βR_F153A_F343A converted progesterone much better than the wild-type enzyme but almost lost its capability of reducing 2-cyclohexen-1-one. Recombinant <i>Draba aizoides</i> P5βR (r<i>Da</i>P5βR) has a second pair of phenylalanines at position 156 and 345 at the rim of the binding site. These two phenylalanines were introduced into r<i>At</i>P5βR_F153A_F343A and the resulting quadruple mutant r<i>At</i>P5βR_F153A_F343A_V156F_V345F partly recovered the ability to reduce 2-cyclohexen-1-one. These results can best be explained by assuming a trapping mechanism in which phenylalanines at the rim of the substrate-binding pocket are involved. The dynamic behavior of individual P5βRs and mutants thereof was investigated by molecular dynamics (MD) simulations and all calculations supported the ‘gatekeeper’ role of phenylalanines periphery of the substrate-binding pocket. Our findings provide structural and mechanistic explanations for the different substrate preferences seen among the natural PRISEs and help to explain the large differences in catalytic efficiency found for different types of 1,4-enones.
DOI: 10.6084/m9.figshare.1568565.v3
title: Pharmacological inhibition of lysosomes activates the MTORC1 signaling pathway in chondrocytes in an autophagy-independent manner

description: Mechanistic target of rapamycin (serine/threonine kinase) complex 1 (MTORC1) is a protein-signaling complex at the fulcrum of anabolic and catabolic processes, which acts depending on wide-ranging environmental cues. It is generally accepted that lysosomes facilitate MTORC1 activation by generating an internal pool of amino acids. Amino acids activate MTORC1 by stimulating its translocation to the lysosomal membrane where it forms a super-complex involving the lysosomal-membrane-bound vacuolar-type H<sup>+</sup>-ATPase (v-ATPase) proton pump. This translocation and MTORC1 activation require functional lysosomes. Here we found that, in contrast to this well-accepted concept, in epiphyseal chondrocytes inhibition of lysosomal activity by v-ATPase inhibitors bafilomycin A<sub>1</sub> or concanamycin A potently activated MTORC1 signaling. The activity of MTORC1 was visualized by phosphorylated forms of RPS6 (ribosomal protein S6) and EIF4EBP1, 2 well-known downstream targets of MTORC1. Maximal RPS6 phosphorylation was observed at 48-h treatment and reached as high as a 12-fold increase (p &lt; 0.018). This activation of MTORC1 was further confirmed in bone organ culture and promoted potent stimulation of longitudinal growth (p &lt; 0.001). Importantly, the same effect was observed in ATG5 (autophagy-related 5)-deficient bones suggesting a macroautophagy-independent mechanism of MTORC1 inhibition by lysosomes. Thus, our data show that in epiphyseal chondrocytes lysosomes inhibit MTORC1 in a macroautophagy-independent manner and this inhibition likely depends on v-ATPase activity.
DOI: 10.6084/m9.figshare.1568551.v2
title: The crucial role of Activin A on the formation of primordial germ cell-like cells from skin-derived stem cells in vitro

description: Primordial germ cells (PGCs) are founder cells of the germ cell lineage, and can be differentiated from stem cells in an induced system <i>in vitro</i>. However, the induction conditions need to be optimized in order to improve the differentiation efficiency. Activin A (ActA) is a member of the TGF-β super family and plays an important role in oogenesis and folliculogenesis. In the present study, we found that ActA promoted PGC-like cells (PGCLCs) formation from mouse skin-derived stem cells (SDSCs) in both embryoid body-like structure (EBLS) differentiation and the co-culture stage in a dose dependent manner. ActA treatment (100 ng/ml) during EBLS differentiation stage and further co-cultured for 6 days without ActA significantly increased PGCLCs from 53.2% to 82.8%, and as well as EBLS differentiation without ActA followed by co-cultured with 100 ng/ml ActA for 4 to 12 days with the percentage of PGCLCs increasing markedly <i>in vitro</i>. Moreover, mice treated with ActA at 100 ng/kg body weight from embryonic day (E) 5.5–12.5 led to more PGCs formation. However, the stimulating effects of ActA were interrupted by <i>Smad3</i> RNAi, and in an <i>in vitro</i> cultured Smad3<sup>−/−</sup> mouse skin cells scenario. SMAD3 is thus likely a key effecter molecule in the ActA signaling pathway. In addition, we found that the expression of some epiblast cell markers, <i>Fgf5, Dnmt3a, Dnmt3b</i> and <i>Wnt3</i>, was increased in EBLSs cultured for 4 days or PGCLCs co-cultured for 12 days with ActA treatment. Interestingly, at 16 days of differentiation, the percentage of PGCLCs was decreased in the presence of ActA, but the expression of meiosis-relative genes, such as <i>Stra8, Dmc1, Sycp3</i> and <i>Sycp1</i>, was increased. In conclusion, our data here demonstrated that ActA can promote PGCLC formation from SDSCs <i>in vitro</i>, at early stages of differentiation, and affect meiotic initiation of PGCLCs in later stages
DOI: 10.6084/m9.figshare.1568551.v1
title: The crucial role of Activin A on the formation of primordial germ cell-like cells from skin-derived stem cells <i>in vitro</i>

description: Primordial germ cells (PGCs) are founder cells of the germ cell lineage, and can be differentiated from stem cells in an induced system <i>in vitro</i>. However, the induction conditions need to be optimized in order to improve the differentiation efficiency. Activin A (ActA) is a member of the TGF-β super family and plays an important role in oogenesis and folliculogenesis. In the present study, we found that ActA promoted PGC-like cells (PGCLCs) formation from mouse skin-derived stem cells (SDSCs) in both embryoid body-like structure (EBLS) differentiation and the co-culture stage in a dose dependent manner. ActA treatment (100 ng/ml) during EBLS differentiation stage and further co-cultured for 6 days without ActA significantly increased PGCLCs from 53.2% to 82.8%, and as well as EBLS differentiation without ActA followed by co-cultured with 100 ng/ml ActA for 4 to 12 days with the percentage of PGCLCs increasing markedly <i>in vitro</i>. Moreover, mice treated with ActA at 100 ng/kg body weight from embryonic day (E) 5.5 – 12.5 led to more PGCs formation. However, the stimulating effects of ActA were interrupted by <i>Smad3</i> RNAi, and in an <i>in vitro</i> cultured Smad3<sup>−/−</sup> mouse skin cells scenario. SMAD3 is thus likely a key effecter molecule in the ActA signaling pathway. In addition, we found that the expression of some epiblast cell markers, <i>Fgf5, Dnmt3a, Dnmt3b</i> and <i>Wnt3</i>, was increased in EBLSs cultured for 4 days or PGCLCs co-cultured for 12 days with ActA treatment. Interestingly, at 16 days of differentiation, the percentage of PGCLCs was decreased in the presence of ActA, but the expression of meiosis-relative genes, such as <i>Stra8, Dmc1, Sycp3</i> and <i>Sycp1</i>, was increased. In conclusion, our data here demonstrated that ActA can promote PGCLC formation from SDSCs <i>in vitro</i>, at early stages of differentiation, and affect meiotic initiation of PGCLCs in later stages.
DOI: 10.6084/m9.figshare.1568539
title: The onset of the Early Eocene Climatic Optimum at Branch Stream, Clarence River valley, New Zealand

description: We present new lithologic, biostratigraphic and carbon isotope records for a calcareous-rich ∼84m thick, early Eocene, upper continental slope section now exposed along Branch Stream, Marlborough. Decimetre-scale limestone-marl couplets comprise the section. Several marl-rich intervals correspond to carbon isotope excursions (CIEs) representing increased <sup>13</sup>C -depleted carbon fluxes to the ocean. These records are similar to those at nearby Mead Stream, except marl-rich intervals at Branch Stream are thicker with a wider δ<sup>13</sup>C range. Comparison to other sites indicates the section spans ∼53.4–51.6 Ma, the onset of the Early Eocene Climatic Optimum (EECO). The most prominent CIE is correlated with the K/X event (52.9 Ma). Prominent marl-rich intervals resulted from increased fluxes of terrigenous material and associated carbonate dilution. We find multiple warming events marked lowermost EECO, each probably signaling enhanced seasonal precipitation. Branch Stream bulk isotopic records suggest 'differential diagenesis' impacted the sequence during sediment burial.
DOI: 10.6084/m9.figshare.1568539.v2
title: The onset of the Early Eocene Climatic Optimum at Branch Stream, Clarence River valley, New Zealand

description: We present new lithologic, biostratigraphic and carbon isotope records for a calcareous-rich ∼84m thick, early Eocene, upper continental slope section now exposed along Branch Stream, Marlborough. Decimetre-scale limestone-marl couplets comprise the section. Several marl-rich intervals correspond to carbon isotope excursions (CIEs) representing increased <sup>13</sup>C -depleted carbon fluxes to the ocean. These records are similar to those at nearby Mead Stream, except marl-rich intervals at Branch Stream are thicker with a wider δ<sup>13</sup>C range. Comparison to other sites indicates the section spans ∼53.4–51.6 Ma, the onset of the Early Eocene Climatic Optimum (EECO). The most prominent CIE is correlated with the K/X event (52.9 Ma). Prominent marl-rich intervals resulted from increased fluxes of terrigenous material and associated carbonate dilution. We find multiple warming events marked lowermost EECO, each probably signaling enhanced seasonal precipitation. Branch Stream bulk isotopic records suggest 'differential diagenesis' impacted the sequence during sediment burial.
DOI: 10.6084/m9.figshare.1568539.v1
title: The onset of the Early Eocene Climatic Optimum at Branch Stream, Clarence River valley, New Zealand

description: We present new lithologic, biostratigraphic and carbon isotope records for a calcareous-rich ∼84m thick, early Eocene, upper continental slope section now exposed along Branch Stream, Marlborough. Decimetre-scale limestone-marl couplets comprise the section. Several marl-rich intervals correspond to carbon isotope excursions (CIEs) representing increased <sup>13</sup>C -depleted carbon fluxes to the ocean. These records are similar to those at nearby Mead Stream, except marl-rich intervals at Branch Stream are thicker with a wider δ<sup>13</sup>C range. Comparison to other sites indicates the section spans ∼53.4–51.6 Ma, the onset of the Early Eocene Climatic Optimum (EECO). The most prominent CIE is correlated with the K/X event (52.9 Ma). Prominent marl-rich intervals resulted from increased fluxes of terrigenous material and associated carbonate dilution. We find multiple warming events marked lowermost EECO, each probably signaling enhanced seasonal precipitation. Branch Stream bulk isotopic records suggest 'differential diagenesis' impacted the sequence during sediment burial.