Drosophila Muller F Elements Maintain a Distinct Set of Genomic Properties Over 40 Million Years of Evolution

The Muller F element (4.2 Mb, similar to 80 protein-coding genes) is an unusual autosome of Drosophila melanogaster; it is mostly heterochromatic with a low recombination rate. To investigate how these properties impact the evolution of repeats and genes, we manually improved the sequence and annotated the genes on the D. erecta, D. mojavensis, and D. grimshawi F elements and euchromatic domains from the Muller D element. We find that F elements have greater transposon density (25-50%) than euchromatic reference regions (3-11%). Among the F elements, D. grimshawi has the lowest transposon density (particularly DINE-1: 2% vs. 11-27%). F element genes have larger coding spans, more coding exons, larger introns, and lower codon bias. Comparison of the Effective Number of Codons with the Codon Adaptation Index shows that, in contrast to the other species, codon bias in D. grimshawi F element genes can be attributed primarily to selection instead of mutational biases, suggesting that density and types of transposons affect the degree of local heterochromatin formation. F element genes have lower estimated DNA melting temperatures than D element genes, potentially facilitating transcription through heterochromatin. Most F element genes (similar to 90%) have remained on that element, but the F element has smaller syntenic blocks than genome averages (3.4-3.6 vs. 8.4-8.8 genes per block), indicating greater rates of inversion despite lower rates of recombination. Overall, the F element has maintained characteristics that are distinct from other autosomes in the Drosophila lineage, illuminating the constraints imposed by a heterochromatic milieu

Head-to-head trial of pegunigalsidase alfa versus agalsidase beta in patients with Fabry disease and deteriorating renal function: results from the 2-year randomised phase III BALANCE study

BACKGROUND: Pegunigalsidase alfa is a PEGylated α-galactosidase A enzyme replacement therapy. BALANCE (NCT02795676) assessed non-inferiority of pegunigalsidase alfa versus agalsidase beta in adults with Fabry disease with an annualised estimated glomerular filtration rate (eGFR) slope more negative than -2 mL/min/1.73 m2/year who had received agalsidase beta for ≥1 year. METHODS: Patients were randomly assigned 2:1 to receive 1 mg/kg pegunigalsidase alfa or agalsidase beta every 2 weeks for 2 years. The primary efficacy analysis assessed non-inferiority based on median annualised eGFR slope differences between treatment arms. RESULTS: Seventy-seven patients received either pegunigalsidase alfa (n=52) or agalsidase beta (n=25). At baseline, mean (range) age was 44 (18-60) years, 47 (61%) patients were male, median eGFR was 74.5 mL/min/1.73 m2 and median (range) eGFR slope was -7.3 (-30.5, 6.3) mL/min/1.73 m2/year. At 2 years, the difference between median eGFR slopes was -0.36 mL/min/1.73 m2/year, meeting the prespecified non-inferiority margin. Minimal changes were observed in lyso-Gb3 concentrations in both treatment arms at 2 years. Proportions of patients experiencing treatment-related adverse events and mild or moderate infusion-related reactions were similar in both groups, yet exposure-adjusted rates were 3.6-fold and 7.8-fold higher, respectively, with agalsidase beta than pegunigalsidase alfa. At the end of the study, neutralising antibodies were detected in 7 out of 47 (15%) pegunigalsidase alfa-treated patients and 6 out of 23 (26%) agalsidase beta-treated patients. There were no deaths. CONCLUSIONS: Based on rate of eGFR decline over 2 years, pegunigalsidase alfa was non-inferior to agalsidase beta. Pegunigalsidase alfa had lower rates of treatment-emergent adverse events and mild or moderate infusion-related reactions. TRIAL REGISTRATION NUMBER: NCT02795676

Drosophila Muller F Elements Maintain a Distinct Set of Genomic Properties Over 40 Million Years of Evolution

The Muller F element (4.2 Mb, ~80 protein-coding genes) is an unusual autosome of Drosophila melanogaster; it is mostly heterochromatic with a low recombination rate. To investigate how these properties impact the evolution of repeats and genes, we manually improved the sequence and annotated the genes on the D. erecta, D. mojavensis, and D. grimshawi F elements and euchromatic domains from the Muller D element. We find that F elements have greater transposon density (25–50%) than euchromatic reference regions (3–11%). Among the F elements, D. grimshawi has the lowest transposon density (particularly DINE-1: 2% vs. 11–27%). F element genes have larger coding spans, more coding exons, larger introns, and lower codon bias. Comparison of the Effective Number of Codons with the Codon Adaptation Index shows that, in contrast to the other species, codon bias in D. grimshawi F element genes can be attributed primarily to selection instead of mutational biases, suggesting that density and types of transposons affect the degree of local heterochromatin formation. F element genes have lower estimated DNA melting temperatures than D element genes, potentially facilitating transcription through heterochromatin. Most F element genes (~90%) have remained on that element, but the F element has smaller syntenic blocks than genome averages (3.4–3.6 vs. 8.4–8.8 genes per block), indicating greater rates of inversion despite lower rates of recombination. Overall, the F element has maintained characteristics that are distinct from other autosomes in the Drosophila lineage, illuminating the constraints imposed by a heterochromatic milieu

The Sequencing, Annotation and Comparative Analysis of the Drosophila mojavensis Dot Chromosome

Mentor: Sarah Elgin From the Washington University Undergraduate Research Digest: WUURD, Volume 7, Issue 1, Fall 2011. Published by the Office of Undergraduate Research, Joy Zalis Kiefer Director of Undergraduate Research and Assistant Dean in the College of Arts & Sciences; Kristin Sobotka, Editor

Novel Approaches in Bottom-Up Proteomic Sample Preparation, Acquisition, and Analysis

The use of proteomic mass spectrometry has become a pervasive component of modern biological and biochemical research. The experimental detection and quantitation of proteins is largely accomplished via liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). In this work, we explore approaches to common problems which pervade three core facets of contemporary proteomic biochemistry by LC-MS/MS: sample preparation, data acquisition, and bioinformatic analysis workflow management.A common step in the sample-preparatory framework is the affinity purification of protein targets, often through the use of antibodies or the protein streptavidin. Avidin proteins such as streptavidin are capable of binding the small molecule biotin with high affinity and specificity. The binding of biotin to streptavidin is oft exploited due to the extremely high affinity and near irreversibility of the interaction. Elution of biotinylated proteins remains inefficient, and many rely on enzymatic digestion, ultimately releasing a large amount of contaminating streptavidin peptides. We explore a method of chemical derivatization which protects streptavidin from tryptic proteolysis, dramatically reducing sample contamination while retaining biotin binding. The method appears generalizable to immunoglobulins antibodies like those against the hemagglutinin epitope.Relative quantitation of proteins and peptides is often performed by comparing the intensities of many samples in a single chromatographic run through multiplexing provided by isobaric tagging reagents. Quantitation of these isobaric tags is observed after fragmentation of a purified analyte, typically selected by Data Dependent Acquisition in a semi-stochastic manner. We explore a new method of acquisition, Sequential Windowed Acquisition of Reporter Masses (SWARM), a Data Independent Acquisition-like approach to isobaric tagged peptide quantitation. This approach biases machine acquisition toward analytes based on their quantitative trends, allowing biologists to focus instrument time on putative analytes of interest.Data produced from the multitude of proteomic experiments must be rigorously analyzed to deconvolute the complex aggregate of mass signals before returning actionable interpretation. The expansion of computational tools the for interrogation of LC-MS/MS data has been a boon to the field, and has made many sophisticated and statistically robust analyses available. However, these tools have been left in unfortunately disjointed sets of software packages lacking convenient interoperability. To help address this problem, we created MilkyWay. MilkyWay is a label free proteomic data analysis platform for quantitative comparisons. Powered by an assemblage of utilities wrapped into the Galaxy bioinformatic workflow management system, MilkyWay contains a R/Shiny web application for the interactive definition of experimental design, file upload, and data exploration

Novel Approaches in Bottom-Up Proteomic Sample Preparation, Acquisition, and Analysis

The use of proteomic mass spectrometry has become a pervasive component of modern biological and biochemical research. The experimental detection and quantitation of proteins is largely accomplished via liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). In this work, we explore approaches to common problems which pervade three core facets of contemporary proteomic biochemistry by LC-MS/MS: sample preparation, data acquisition, and bioinformatic analysis workflow management.A common step in the sample-preparatory framework is the affinity purification of protein targets, often through the use of antibodies or the protein streptavidin. Avidin proteins such as streptavidin are capable of binding the small molecule biotin with high affinity and specificity. The binding of biotin to streptavidin is oft exploited due to the extremely high affinity and near irreversibility of the interaction. Elution of biotinylated proteins remains inefficient, and many rely on enzymatic digestion, ultimately releasing a large amount of contaminating streptavidin peptides. We explore a method of chemical derivatization which protects streptavidin from tryptic proteolysis, dramatically reducing sample contamination while retaining biotin binding. The method appears generalizable to immunoglobulins antibodies like those against the hemagglutinin epitope.Relative quantitation of proteins and peptides is often performed by comparing the intensities of many samples in a single chromatographic run through multiplexing provided by isobaric tagging reagents. Quantitation of these isobaric tags is observed after fragmentation of a purified analyte, typically selected by Data Dependent Acquisition in a semi-stochastic manner. We explore a new method of acquisition, Sequential Windowed Acquisition of Reporter Masses (SWARM), a Data Independent Acquisition-like approach to isobaric tagged peptide quantitation. This approach biases machine acquisition toward analytes based on their quantitative trends, allowing biologists to focus instrument time on putative analytes of interest.Data produced from the multitude of proteomic experiments must be rigorously analyzed to deconvolute the complex aggregate of mass signals before returning actionable interpretation. The expansion of computational tools the for interrogation of LC-MS/MS data has been a boon to the field, and has made many sophisticated and statistically robust analyses available. However, these tools have been left in unfortunately disjointed sets of software packages lacking convenient interoperability. To help address this problem, we created MilkyWay. MilkyWay is a label free proteomic data analysis platform for quantitative comparisons. Powered by an assemblage of utilities wrapped into the Galaxy bioinformatic workflow management system, MilkyWay contains a R/Shiny web application for the interactive definition of experimental design, file upload, and data exploration

Atlas of metabolic diseases / William L. Nyhan , Bruce A. Barshop , Pinar T. Ozand.

Includes bibliographical references and indexes.xii, 788 pages

Cell Surface Proteomics Provides Insight into Stage-Specific Remodeling of the Host-Parasite Interface in Trypanosoma brucei*

African trypanosomes are devastating human and animal pathogens transmitted by tsetse flies between mammalian hosts. The trypanosome surface forms a critical host interface that is essential for sensing and adapting to diverse host environments. However, trypanosome surface protein composition and diversity remain largely unknown. Here, we use surface labeling, affinity purification, and proteomic analyses to describe cell surface proteomes from insect-stage and mammalian bloodstream-stage Trypanosoma brucei. The cell surface proteomes contain most previously characterized surface proteins. We additionally identify a substantial number of novel proteins, whose functions are unknown, indicating the parasite surface proteome is larger and more diverse than generally appreciated. We also show stage-specific expression for individual paralogs within several protein families, suggesting that fine-tuned remodeling of the parasite surface allows adaptation to diverse host environments, while still fulfilling universally essential cellular needs. Our surface proteome analyses complement existing transcriptomic, proteomic, and in silico analyses by highlighting proteins that are surface-exposed and thereby provide a major step forward in defining the host-parasite interface

Iron-regulated assembly of the cytosolic iron-sulfur cluster biogenesis machinery.

The cytosolic iron-sulfur (Fe-S) cluster assembly (CIA) pathway delivers Fe-S clusters to nuclear and cytosolic Fe-S proteins involved in essential cellular functions. Although the delivery process is regulated by the availability of iron and oxygen, it remains unclear how CIA components orchestrate the cluster transfer under varying cellular environments. Here, we utilized a targeted proteomics assay for monitoring CIA factors and substrates to characterize the CIA machinery. We find that nucleotide-binding protein 1 (NUBP1/NBP35), cytosolic iron-sulfur assembly component 3 (CIAO3/NARFL), and CIA substrates associate with nucleotide-binding protein 2 (NUBP2/CFD1), a component of the CIA scaffold complex. NUBP2 also weakly associates with the CIA targeting complex (MMS19, CIAO1, and CIAO2B) indicating the possible existence of a higher order complex. Interactions between CIAO3 and the CIA scaffold complex are strengthened upon iron supplementation or low oxygen tension, while iron chelation and reactive oxygen species weaken CIAO3 interactions with CIA components. We further demonstrate that CIAO3 mutants defective in Fe-S cluster binding fail to integrate into the higher order complexes. However, these mutants exhibit stronger associations with CIA substrates under conditions in which the association with the CIA targeting complex is reduced suggesting that CIAO3 and CIA substrates may associate in complexes independently of the CIA targeting complex. Together, our data suggest that CIA components potentially form a metabolon whose assembly is regulated by environmental cues and requires Fe-S cluster incorporation in CIAO3. These findings provide additional evidence that the CIA pathway adapts to changes in cellular environment through complex reorganization