Visualisation of experimentally determined and predicted protein N-glycosylation and predicted glycosylphosphatidylinositol anchor addition in Trypanosoma brucei.

Background: Trypanosoma brucei is a protozoan parasite and the etiological agent of human and animal African trypanosomiasis. The organism cycles between its mammalian host and tsetse vector. The host-dwelling bloodstream form of the parasite is covered with a monolayer of variant surface glycoprotein (VSG) that enables it to escape both the innate and adaptive immune systems. Within this coat reside lower-abundance surface glycoproteins that function as receptors and/or nutrient transporters. The glycosylation of the Trypanosoma brucei surface proteome is essential to evade the immune response and is mediated by three oligosaccharyltransferase genes; two of which, TbSTT3A and TbSTT3B, are expressed in the bloodstream form of the parasite. Methods: We processed a recent dataset of our laboratory to visualise putative glycosylation sites of the Trypanosoma brucei proteome. We provided a visualisation for the predictions of glycosylation carried by TbSTT3A and TbSTT3B, and we augmented the visualisation with predictions for Glycosylphosphatidylinositol anchoring sites, domains and topology of the Trypanosoma brucei proteome. Conclusions: We created a web service to explore the glycosylation sites of the Trypanosoma brucei oligosaccharyltransferases substrates, using data described in a recent publication of our laboratory. We also made a machine learning algorithm available as a web service, described in our recent publication, to distinguish between TbSTT3A and TbSTT3B substrates

Visualisation of proteome-wide ordered protein abundances in Trypanosoma brucei

Background: Trypanosoma brucei is a protozoan parasite and etiological agent of human and animal African trypanosomiasis. It has a complex life cycle, but the most studied cellular types are the in vitro cultivated bloodstream- and procyclic-forms. These correspond to the replicating, mammalian host bloodstream-dwelling, slender trypomastigotes and tsetse vector midgut-dwelling procyclic lifecycle stages, respectively. Several proteomics studies have reported the differential abundance of proteins between these in vitro cultivated cell types. However, there are no datasets providing protein abundance, from most to least abundant, within and between both cell types. Methods: We used MaxQuant software 1.6.10.4 to reprocess a recent large-scale proteomics experiment from our laboratory and extracted intensity-based quantifications of the bloodstream and procyclic form proteomes. Results: We created a web interface to visually explore protein abundances within and between the in vitro cultivated T. brucei bloodstream and procyclic form proteomes. Conclusions: The protein abundance visualization tool, searchable by protein name(s) and attribute(s), is likely to be useful to the trypanosome research community. It will allow users to contextualise their proteins of interest in terms of their abundances in the T. brucei bloodstream and procyclic form proteomes

Common and unique features of glycosylation and glycosyltransferases in African trypanosomes

Eukaryotic protein glycosylation is mediated by glycosyl- and oligosaccharyl-transferases. Here, we describe how African trypanosomes exhibit both evolutionary conservation and significant divergence compared with other eukaryotes in how they synthesise their glycoproteins. The kinetoplastid parasites have conserved components of the dolichol-cycle and oligosaccharyltransferases (OSTs) of protein N-glycosylation, and of glycosylphosphatidylinositol (GPI) anchor biosynthesis and transfer to protein. However, some components are missing, and they process and decorate their N-glycans and GPI anchors in unique ways. To do so, they appear to have evolved a distinct and functionally flexible glycosyltransferases (GT) family, the GT67 family, from an ancestral eukaryotic β3GT gene. The expansion and/or loss of GT67 genes appears to be dependent on parasite biology. Some appear to correlate with the obligate passage of parasites through an insect vector, suggesting they were acquired through GT67 gene expansion to assist insect vector (tsetse fly) colonisation. Others appear to have been lost in species that subsequently adopted contaminative transmission. We also highlight the recent discovery of a novel and essential GT11 family of kinetoplastid parasite fucosyltransferases that are uniquely localised to the mitochondria of Trypanosoma brucei and Leishmania major. The origins of these kinetoplastid FUT1 genes, and additional putative mitochondrial GT genes, are discussed

Proteomic identification of the UDP-GlcNAc:PI α1-6 GlcNAc-transferase subunits of the glycosylphosphatidylinositol biosynthetic pathway of <i>Trypanosoma brucei</i>

The first step of glycosylphosphatidylinositol (GPI) anchor biosynthesis in all eukaryotes is the addition of N-acetylglucosamine (GlcNAc) to phosphatidylinositol (PI) which is catalysed by a UDP-GlcNAc: PI α1-6 GlcNAc-transferase, also known as GPI GnT. This enzyme has been shown to be a complex of seven subunits in mammalian cells and a similar complex of six homologous subunits has been postulated in yeast. Homologs of these mammalian and yeast subunits were identified in the Trypanosoma brucei predicted protein database. The putative catalytic subunit of the T. brucei complex, TbGPI3, was epitope tagged with three consecutive c-Myc sequences at its C-terminus. Immunoprecipitation of TbGPI3-3Myc followed by native polyacrylamide gel electrophoresis and anti-Myc Western blot showed that it is present in a ~240 kDa complex. Label-free quantitative proteomics were performed to compare anti-Myc pull-downs from lysates of TbGPI-3Myc expressing and wild type cell lines. TbGPI3-3Myc was the most highly enriched protein in the TbGPI3-3Myc lysate pull-down and the expected partner proteins TbGPI15, TbGPI19, TbGPI2, TbGPI1 and TbERI1 were also identified with significant enrichment. Our proteomics data also suggest that an Arv1-like protein (TbArv1) is a subunit of the T. brucei complex. Yeast and mammalian Arv1 have been previously implicated in GPI biosynthesis, but here we present the first experimental evidence for physical association of Arv1 with GPI biosynthetic machinery. A putative E2-ligase has also been tentatively identified as part of the T. brucei UDP-GlcNAc: PI α1-6 GlcNAc-transferase complex

The PAndAS view of the Andromeda satellite system - I. A Bayesian search for dwarf galaxies using spatial and color-magnitude information

We present a generic algorithm to search for dwarf galaxies in photometric catalogs and apply it to the Pan-Andromeda Archaeological Survey (PAndAS). The algorithm is developed in a Bayesian framework and, contrary to most dwarf-galaxy-search codes, makes use of both the spatial and color-magnitude information of sources in a probabilistic approach. Accounting for the significant contamination from the Milky Way foreground and from the structured stellar halo of the Andromeda galaxy, we recover all known dwarf galaxies in the PAndAS footprint with high significance, even for the least luminous ones. Some Andromeda globular clusters are also recovered and, in one case, discovered. We publish a list of the 143 most significant detections yielded by the algorithm. The combined properties of the 39 most significant isolated detections show hints that at least some of these trace genuine dwarf galaxies, too faint to be individually detected. Follow-up observations by the community are mandatory to establish which are real members of the Andromeda satellite system. The search technique presented here will be used in an upcoming contribution to determine the PAndAS completeness limits for dwarf galaxies. Although here tuned to the search of dwarf galaxies in the PAndAS data, the algorithm can easily be adapted to the search for any localised overdensity whose properties can be modeled reliably in the parameter space of any catalog.Comment: 20 pages, 16 figures, 1 table; accepted for publication in ApJ. High res pdf available at https://www.dropbox.com/s/7zk7pme2wunwkjv/PAndAS_dwarf_galaxies.pd

7,7′-(3,3′-Dibenzyl-3H,3′H-4,4′-bi-1,2,3-triazole-5,5′-di­yl)bis­(4-methyl-2H-chromen-2-one)

The title compound, a bis-5,5′-triazole, C38H28N6O4, was observed as a side-product from the Sharpless–Meldal click reaction of the corresponding coumarin alkyne and benzyl­azide. Although the compound was present as a minor component, it crystallized in preference to the major product. The two triazole rings are almost orthogonal to each other [dihedral angle = 83.8 (1)°]. However the 4 and 4′ coumarin systems are close to coplanar with their respective triazole rings [23.6 (1) and 15.1 (1)°]. Each of the benzene rings packs approximately face-to-face with the opposing coumarin ring systems, with inter­planar angles of 7.7 (1) and 25.3 (1)° and distances of 3.567 (2) and 3.929 (2) Å between the respective centroids of the opposing rings

N-glycan microheterogeneity regulates interactions of plasma proteins

Altered glycosylation patterns of plasma proteins are associated with autoimmune disorders and pathogenesis of various cancers. Elucidating glycoprotein microheterogeneity and relating subtle changes in the glycan structural repertoire to changes in protein–protein, or protein–small molecule interactions, remains a significant challenge in glycobiology. Here, we apply mass spectrometry-based approaches to elucidate the global and site-specific microheterogeneity of two plasma proteins: α1-acid glycoprotein (AGP) and haptoglobin (Hp). We then determine the dissociation constants of the anticoagulant warfarin to different AGP glycoforms and reveal how subtle N-glycan differences, namely, increased antennae branching and terminal fucosylation, reduce drug-binding affinity. Conversely, similar analysis of the haptoglobin–hemoglobin (Hp–Hb) complex reveals the contrary effects of fucosylation and N-glycan branching on Hp–Hb interactions. Taken together, our results not only elucidate how glycoprotein microheterogeneity regulates protein–drug/protein interactions but also inform the pharmacokinetics of plasma proteins, many of which are drug targets, and whose glycosylation status changes in various disease states

A longitudinal study of multicultural curriculum in medical education

OBJECTIVE: To evaluate impact a multicultural interclerkship had on students\u27 perception of knowledge, interview skills, and empathy towards serving culturally diverse populations and role student demographics played in learning. METHODS: Data extracted from students\u27 self-reported course evaluations and pre/post questionnaires during multiculturalism interclerkship across 11 academic years. Inquired students\u27 opinion about four areas: effectiveness, small group leaders, usefulness, and overall experience. Subscale and item ratings were compared using trend tests including multivariate analyses. RESULTS: During studied years, 883 students completed course evaluation with high overall mean rating of 3.08 (S = 0.45) and subscale mean scores ranging from 3.03 to 3.30. Trends in three of four subscales demonstrated clear uptrend (p \u3c 0.0001). Positive correlations between ratings of leaders and usefulness were observed (p \u3c 0.0001). Pre/post matched dataset (n = 967) indicated majority of items (19/23) had statistically significant higher post interclerkship ratings compared to pre scores with nine of 19 having statistically significant magnitudes of change. Questionnaire had high overall reliability (Cronbach alpha = 0.8), and item-to-group correlations ranged from 0.40 to 0.68 (p \u3c 0.0001). CONCLUSIONS: By increasing students\u27 exposure and interaction with diverse patients, their knowledge, attitude, and skills were increased and expanded in positive manner. These findings might inform those who are interested in enhancing this important competence. This is especially true given increasing scrutiny this global topic is receiving within and across healthcare professions around the world

Faint AGN and the Ionizing Background

We determine the evolution of the faint, high-redshift, optical luminosity function (LF) of AGN implied by several observationally-motivated models of the ionizing background. Our results depend crucially on whether we use the total ionizing rate measured by the proximity effect technique or the lower determination from the flux decrement distribution of Ly alpha forest lines. Assuming a faint-end LF slope of 1.58 and the SDSS estimates of the bright-end slope and normalization, we find that the LF must break at M_B*=-24.2,-22.3, -20.8 at z=3,4, 5 if we adopt the lower ionization rate and assume no stellar contribution to the background. The break must occur at M_B*=-20.6,-18.7, -18.7 for the proximity effect background estimate. These values brighten by as much as ~2 mag if high-z galaxies contribute to the background with an escape fraction of ionizing photons consistent with recent estimates: f_e=0.16. By comparing to faint AGN searches, we find that the typically-quoted proximity effect estimates of the background imply an over-abundance of faint AGN (even with f_e=1). Even adopting the lower bound on proximity effect measurements, the stellar escape fraction must be high: f_e>0.2. Conversely, the lower flux- decrement-derived background requires a limited stellar contribution: f_e<0.05. Our derived LFs together with the locally-estimated black hole density suggest that the efficiency of converting mass to light in optically-unobscured AGN is somewhat lower than expected, <0.05. Comparison with similar estimates based on X-ray counts suggests that more than half of all AGN are obscured in the UV/optical. We also derive lower limits on typical AGN lifetimes and obtain >10^7 yrs for favored cases.Comment: 19 pages, 16 figures. Accepted by Astrophysical Journa