585 research outputs found
Use of isotopically labeled substrates reveals kinetic differences between human and bacterial serine palmitoyltransferase
Isotope labels are frequently used tools to track metabolites through complex biochemical pathways and to discern the mechanisms of enzyme-catalysed reactions. Isotopically-labelled L-serine is often used to monitor the activity of the first enzyme in sphingolipid biosynthesis, serine palmitoyltransferase (SPT) as well as labelling downstream cellular metabolites. Intrigued by the effect that isotope labels may be having on SPT catalysis, we characterised the impact of different L-serine isotopologues on the catalytic activity of recombinant SPT isozymes from humans and the bacterium Sphingomonas paucimobilis. Our data show that S. paucimobilis SPT activity displays a clear isotope effect with [2,3,3-D] L-serine, whereas the human SPT isoform does not. This suggests that whilst both human and S. paucimobilis SPT catalyse the same chemical reaction, there may well be underlying subtle differences in their catalytic mechanisms. Our results suggest that it is that the activating small subunits of human SPT that play a key role in these mechanistic variations. This study also highlight that it is important to consider the type and location of isotope labels on a substrate when they are to be used in in vitro and in vivo studies
Determination of protein thiol reduction potential by isotope labeling and intact mass measurement
Oxidation/reduction of thiol residues in proteins is an important type of post-translational modification that is implicated in regulating a range of biological processes. The nature of the modification makes it possible to define a quantifiable electrochemical potential, E⊕, for oxidation/reduction that allows cysteine-containing proteins to be ranked based on their propensity to be oxidized. Measuring oxidation of cysteine residues in proteins is difficult using standard electrochemical methods but recently top-down mass-spectrometry has been shown to enable the quantification of E⊕ for thiol oxidations. In this paper we demonstrate that mass spectrometry of intact proteins can be used in combination with an isotopic labeling strategy and an automated data analysis algorithm to measure E⊕ for the thiols in both E Coli Thioredoxin 1 and Human Thioredoxin 1. Our methodology relies on accurate mass measurement of proteins using LC-MS analyses and does not necessarily require top-down fragmentation. As well as analyzing homogeneous protein samples, we also demonstrate that our methodology can be used to determine thiol E⊕ measurements in samples which contain mixtures of proteins. Thus the combination of experiential methodology and data analysis regime have the potential to make such measurements in a high-throughput manner and in a manner more accessible to a broad community of protein scientists
Characterization of secreted sphingosine-1-phosphate lyases required for virulence and intracellular survival of <i>Burkholderia pseudomallei</i>
This is the author accepted manuscript. The final version is available from the publisher via the DOI in this record.Sphingosine-1-phosphate (S1P), a bioactive sphingolipid metabolite, plays a critical role in the orchestration of immune responses. S1P levels within the mammalian host are tightly regulated, in part through the activity of S1P lyase (S1PL) which catalyses its irreversible degradation. Herein we describe the identification and characterization of secreted S1PL orthologues encoded by the facultative intracellular bacteria Burkholderia pseudomallei and Burkholderia thailandensis. These bacterial orthologues exhibited S1PL enzymatic activity, functionally complemented an S1PL-deficient yeast strain, and conferred resistance to the antimicrobial sphingolipid D-erythro-sphingosine. We report that secretion of these bacterial S1PLs is pH-dependent, and is observed during intracellular infection. S1PL-deficient mutants displayed impaired intracellular replication in murine macrophages (associated with an inability to evade the maturing phagosome) and were significantly attenuated in murine and larval infection models. Furthermore, treatment of Burkholderia-infected macrophages with either S1P or a selective agonist of S1P receptor 1 enhanced bacterial colocalisation with LAMP-1 and reduced their intracellular survival. In summary, our studies confirm bacterial-encoded S1PL as a critical virulence determinant of B. pseudomallei and B. thailandensis, further highlighting the pivotal role of S1P in host-pathogen interactions. In addition, our data suggest that S1P pathway modulators have potential for the treatment of intracellular infection.We thank HL Ho & K Haynes (University of Exeter) for provision of strains and relevant
vectors for yeast complementation studies. This work was supported by the Defence Science
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and Technology Laboratory under contract DSTLX-1000060221 (WP1). CJM was funded by
the EASTBIO Doctoral Training Partnership. The funders had no role in study design, data
collection and analysis, or preparation of the manuscript
Shape analysis of the StW 578 calotte from Jacovec Cavern, Gauteng (South Africa)
The fossiliferous deposits within the lower-lying Jacovec Cavern in the locality of Sterkfontein yielded valuable hominin remains, including the StW 578 specimen. Because StW 578 mainly preserves the calotte, the taxonomic status of this specimen has been a matter of discussion. Within this context, here we employed high-resolution microtomography and a landmark-free registration method to explore taxonomically diagnostic features in the external surface of the StW 578 calotte. Our comparative sample included adult humans and common chimpanzees as well as one Australopithecus africanus specimen (Sts 5). We partially restored the StW 578 calotte digitally and compared it to extant specimens and Sts 5 using a landmark-free registration based on smooth and invertible surface deformation. Our comparative shape analysis reveals morphological differences with extant humans, especially in the frontal bones, and with extant chimpanzees, as well as intriguing specificities in the morphology of the StW 578 parietal bones. Lastly, our study suggests morphological proximity between StW 578 and Sts 5. Given the intimate relationship between the brain and the braincase, as well as the integration of the hominin face and neurocranium, we suggest that cranial vault shape differences between StW 578 and extant humans, if confirmed by further analyses, could be either explained by differences in brain surface morphology or in the face. Besides providing additional information about the morphology of the Jacovec calotte that will be useful in future taxonomic discussion, this study introduces a new protocol for the landmark-free analysis of fossil hominin cranial shape.Significance:• We provide further information on the enigmatic fossil specimen StW 578.• We introduce a new approach for the morphological study of fossil hominin crania.• We highlight morphological similarities between StW 578 and ‘Mrs Ples’
Observation of large spontaneous emission rate enhancement of quantum dots in a broken-symmetry slow-light waveguide
Quantum states of light and matter can be manipulated on the nanoscale to
provide a technological resource for aiding the implementation of scalable
photonic quantum technologies [1-3]. Experimental progress relies on the
quality and efficiency of the coupling between photons and internal states of
quantum emitters [4-6]. Here we demonstrate a nanophotonic waveguide platform
with embedded quantum dots (QDs) that enables both Purcell-enhanced emission
and strong chiral coupling. The design uses slow-light effects in a glide-plane
photonic crystal waveguide with QD tuning to match the emission frequency to
the slow-light region. Simulations were used to map the chirality and Purcell
enhancement depending on the position of a dipole emitter relative to the air
holes. The highest Purcell factors and chirality occur in separate regions, but
there is still a significant area where high values of both can be obtained.
Based on this, we first demonstrate a record large radiative decay rate of 17
ns^-1 (60 ps lifetime) corresponding to a 20 fold Purcell enhancement. This was
achieved by electric-field tuning of the QD to the slow-light region and
quasi-resonant phonon-sideband excitation. We then demonstrate a 5 fold Purcell
enhancement for a dot with high degree of chiral coupling to waveguide modes,
substantially surpassing all previous measurements. Together these demonstrate
the excellent prospects for using QDs in scalable implementations of on-chip
spin-photonics relying on chiral quantum optics.Comment: 15 pages, 4 figures, 1 table. Supporting information is available
upon request to the corresponding autho
Peter Barlow's insights and contributions to the study of tidal gravity variations and ultra-weak light emissions in plants
High prevalence of subclinical vitamin K1Â deficiency among human milk-fed preterm infants in early infancy
Reconstitution of the pyridoxal 5'-phosphate (PLP) dependent enzyme serine palmitoyltransferase (SPT) with pyridoxal reveals a crucial role for the phosphate during catalysis
Redox Chemistry and the Role of Trapped Molecular O<sub>2</sub>in Li-Rich Disordered Rocksalt Oxyfluoride Cathodes
In the search for high energy density cathodes for next-generation lithium-ion batteries, the disordered rocksalt oxyfluorides are receiving significant attention due to their high capacity and lower voltage hysteresis compared with ordered Li-rich layered compounds. However, a deep understanding of these phenomena and their redox chemistry remains incomplete. Using the archetypal oxyfluoride, Li2MnO2F, we show that the oxygen redox process in such materials involves the formation of molecular O2 trapped in the bulk structure of the charged cathode, which is reduced on discharge. The molecular O2 is trapped rigidly within vacancy clusters and exhibits minimal mobility unlike free gaseous O2, making it more characteristic of a solid-like environment. The Mn redox process occurs between octahedral Mn3+ and Mn4+ with no evidence of tetrahedral Mn5+ or Mn7+. We furthermore derive the relationship between local coordination environment and redox potential; this gives rise to the observed overlap in Mn and O redox couples and reveals that the onset potential of oxide ion oxidation is determined by the degree of ionicity around oxygen, which extends models based on linear Li-O-Li configurations. This study advances our fundamental understanding of redox mechanisms in disordered rocksalt oxyfluorides, highlighting their promise as high capacity cathodes
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