We review and demonstrate how an empirical Bayes method, shrinking a protein's sample variance towards pooled estimate, leads to far more powerful stable inference detect significant changes in protein abundance compared ordinary t-tests. Using examples from isobaric mass labeled proteomic experiments we show analyze data multiple simultaneously, discuss the effects of missing on inference. also present easy use open source software for normalization spectrometry based moderated test statistics.

Identification of replication initiation sites, termed origins, is a crucial step in understanding genome transmission any organism.Transcription the Trypanosoma brucei highly unusual, with each chromosome comprising few discrete transcription units.To understand how DNA occurs context such organization, we have performed genome-wide mapping binding sites initiator ORC1/CDC6 and identified revealing that both localize to boundaries units.A remarkably small number active origins seen, whose...

Kinetoplastid parasites cause diseases that threaten human and animal health. To survive transitions between vertebrate hosts insect vectors, these rely on precise regulation of gene expression to adapt environmental changes. Since in kinetoplastids is primarily post-transcriptional, developing efficient genetic tools for modifying genes at their endogenous loci while preserving regulatory mRNA elements crucial studying complex biology. We present a CRISPR/Cas9-based tagging system preserves...

DNA Replication initiates by formation of a pre-replication complex on sequences termed origins. In eukaryotes, the is composed Origin Recognition Complex (ORC), Cdc6 and MCM replicative helicase in conjunction with Cdt1. Eukaryotic ORC considered to be six subunits, named Orc1–6, monomeric closely related sequence Orc1. However, has been little explored protists, only single protein, both Orc1 Cdc6, shown act replication Trypanosoma brucei. Here we identify three highly diverged putative T....

Initiation of DNA replication depends upon recognition genomic sites, termed origins, by AAA+ ATPases. In prokaryotes a single factor binds each origin, whereas in eukaryotes this role is played six-protein origin complex (ORC). Why evolved multisubunit initiator, and the roles component, remains unclear. Trypanosoma brucei, an ancient unicellular eukaryote, only one ORC-related TbORC1/CDC6, has been identified sequence homology. Here we show that three TbORC1/CDC6-interacting factors also...

Variant surface glycoprotein (VSG) is central to antigenic variation in African trypanosomes. Although much prior work documents that VSG efficiently synthesized and exported the cell surface, it was recently claimed 2-3 fold more than required, excess being eliminated by ER-Associated Degradation (ERAD) (Field et al., ). We now reinvestigate turnover find no evidence for rapid degradation, consistent with a model whereby synthesis precisely regulated match requirements functional coat on...

Bloodstream-form African trypanosomes encode two structurally related glycosylphosphatidylinositol (GPI)-anchored proteins that are critical virulence factors, variant surface glycoprotein (VSG) for antigenic variation and transferrin receptor (TfR) iron acquisition. Both transcribed from the active telomeric expression site. VSG is a GPI2 homodimer; TfR GPI1 heterodimer of GPI-anchored ESAG6 ESAG7. GPI-valence correlates with secretory progression fate in bloodstream trypanosomes: (GPI2)...

Summary In an RNAi library screen for loss of kinetoplast DNA ( kDNA ), we identified uncharacterized T rypanosoma brucei protein, named TbLOK1 , required maintenance mitochondrial shape and function. We found the protein located in discrete patches outer membrane. Knock‐down procyclic trypanosomes caused highly interconnected structure to collapse, forming unbranched tubule remarkably similar streamlined organelle seen bloodstream form. Following defects respiration, inner membrane...

Iron is an essential regulatory signal for virulence factors in many pathogens. Mammals and bloodstream form (BSF) Trypanosoma brucei obtain iron by receptor-mediated endocytosis of transferrin bound to receptors (TfR) but the mechanisms which T. subsequently handles remains enigmatic. Here, we analyse transcriptome cultured iron-rich iron-poor conditions. We show that adaptation iron-deprivation induces upregulation TfR, a cohort parasite-specific genes (ESAG3, PAGS), involved glucose...

Misfolded secretory proteins are retained by endoplasmic reticulum quality control (ERQC) and degraded in the proteasome ER-associated degradation (ERAD). However, yeast mammals, misfolded glycosylphosphatidylinositol (GPI)-anchored preferentially vacuole/lysosome. We investigate this process divergent eukaryotic pathogen Trypanosoma brucei using a GPI-anchored subunit (HA:E6) of trypanosome transferrin receptor. HA:E6 is N-glycosylated accumulates ER as aggregates. Treatment with MG132,...

The unfolded protein response (UPR) is a stress mechanism to cope with misfolded proteins in the early secretory pathway, hallmark being transcriptional upregulation of endoplasmic reticulum (ER) molecular chaperones such as BiP and disulfide isomerase. Despite lack regulation absence classical UPR machinery, African trypanosomes apparently respond persistent ER by UPR-like response, including BiP, related spliced leader silencing (SLS) whereby SL RNA transcription shut down. Initially...

Cellular barcoding techniques are powerful tools to understand microbial pathogenesis. However, strategies have not been broadly applied protozoan parasites, which unique genomic structures and virulence compared with viral bacterial pathogens. Here, we present a CRISPR-based method barcode protozoa, successfully apply Toxoplasma gondii Trypanosoma brucei. Using libraries of barcoded T. gondii, evaluate shifts in the population structure from acute chronic infection mice. Contrary...

Abstract Kinetoplastid parasites cause diseases that threaten human and animal health. To survive transitions between vertebrate hosts insect vectors, these rely on precise regulation of gene expression to adapt environmental changes. Since in Kinetoplastids is primarily post-transcriptional, developing efficient genetic tools for modifying genes at their endogenous loci while preserving regulatory mRNA elements crucial studying complex biology. We present a high-throughput CRISPR/Cas9-based...

The transferrin receptor (TfR) of the bloodstream form (BSF) Trypanosoma brucei is a heterodimer comprising glycosylphosphatidylinositol (GPI)-anchored expression site-associated gene 6 (ESAG6 or E6) and soluble ESAG7. Mature E6 has five N-glycans, consisting three oligomannose two unprocessed paucimannose structures. Its GPI anchor modified by addition 4-6 α-galactose residues. TfR binds tomato lectin (TL), specific for N-acetyllactosamine (LacNAc) repeats, previous studies have shown...

The bloodstream form of Trypanosoma brucei expresses large poly- N -acetyllactosamine (pNAL) chains on complex N-glycans a subset glycoproteins. It has been hypothesised that pNAL may be required for receptor-mediated endocytosis. African trypanosomes contain unique family glycosyltransferases, the GT67 family. Two these, TbGT10 and TbGT8, have shown to involved in biosynthesis , raising possibility deleting both enzymes simultaneously might abolish provide clues function and/or...

The bloodstream form of Trypanosoma brucei expresses large poly-N-acetyllactosamine (pNAL) chains on complex N-glycans a subset glycoproteins. It has been hypothesised that pNAL may be required for receptor-mediated endocytosis. African trypanosomes contain unique family glycosyltransferases, the GT67 family. Two these, TbGT10 and TbGT8, have shown to involved in biosynthesis brucei, raising possibility deleting both enzymes simultaneously might abolish provide clues function and/or...

Abstract Molecular barcoding techniques have emerged as powerful tools to understand microbial pathogenesis. However, strategies not been extended protozoan parasites, which unique genomic structures and virulence compared viral bacterial pathogens. Here, we present a versatile CRISPR-based method barcode protozoa, successfully apply Toxoplasma gondii Trypanosoma brucei . The murine brain is an important transmission niche for T. , persistence clinically untreatable feature of infection....

Visualisation of genomic loci by microscopy is essential for understanding nuclear organisation, particularly at the single cell level. One powerful technique studying positioning through Lac Operator-Lac Repressor (LacO-LacI) system, in which LacO repeats introduced into a specific locus can be visualised expression LacI-protein fused to fluorescent tag. First utilised Trypanosoma brucei over 20 years ago, we have now optimised this system with short, stabilised less than 2 kb paired...

Calvin Tiengwe works on posttranscriptional gene regulation and iron homeostasis in the parasitic protozoan Trypanosoma brucei In this mSphere of Influence article, he reflects how paper "Comprehensive identification RNA-protein interactions any organism using orthogonal organic phase separation (OOPS)" by Queiroz et al. (Nat Biotechnol 37:169-178, 2019, https://doi.org/10.1038/s41587-018-0001-2) influenced his research providing a tool to capture complexes global scale acid guanidinium...

Molecular barcoding techniques have emerged as powerful tools to understand microbial pathogenesis. However, strategies not been extended protozoan parasites, which unique genomic structures and virulence compared viral bacterial pathogens. Here, we present a versatile CRISPR-based method barcode protozoa, successfully apply Toxoplasma gondii Trypanosoma brucei. The murine brain is an important transmission niche for T. gondii, persistence clinically untreatable feature of infection....

Abstract Iron is an essential regulatory signal for virulence factors in many pathogens. Mammals and bloodstream form (BSF) Trypanosoma brucei obtain iron by receptor-mediated endocytosis of transferrin bound to receptors (TfR) but the mechanisms which T. subsequently handles remains enigmatic. Here, we analyse transcriptome cultured iron-rich iron-poor conditions. We show that adaptation iron-deprivation induces upregulation TfR, a cohort parasite-specific genes (ESAG3, PAGS), involved...