Structural Basis for the Secretion of EvpC: A Key Type VI Secretion System Protein from Edwardsiella tarda

The recently identified type VI secretion system (T6SS) is implicated in the virulence of many Gram-negative bacteria. Edwardsiella tarda is an important cause of hemorrhagic septicemia in fish and also gastro- and extra-intestinal infections in humans. The E. tarda virulent protein (EVP) gene cluster encodes a conserved T6SS which contains 16 open reading frames. EvpC is one of the three major EVP secreted proteins and shares high sequence similarity with Hcp1, a key T6SS virulence factor from Pseudomonas aeruginosa. EvpC contributes to the virulence of E. tarda by playing an essential role in functional T6SS. Here, we report the crystal structure of EvpC from E. tarda PPD130/91 at a 2.8 Å resolution, along with functional studies of the protein. EvpC has a β-barrel domain with extended loops. The β-barrel consists of 11 anti-parallel β-strands with an α-helix located on one side. In solution, EvpC exists as a dimer at low concentration and as a hexamer at higher concentration. In the crystal, the symmetry related EvpC molecules form hexameric rings which stack together to form a tube similar to Hcp1. Structure based mutagenesis revealed that N-terminal negatively charged residues, Asp4, Glu15 and Glu26, and C-terminal positively charged residues, Lys161, Lys162 and Lys163, played crucial roles in the secretion of EvpC. Moreover, the localization study indicates the presence of wild type EvpC in cytoplasm, periplasm and secreted fractions, whereas the N-terminal and C-terminal mutants were found mostly in the periplasmic region and was completely absent in the secreted fraction. Results reported here provide insight into the structure, assembly and function of EvpC. Further, these findings can be extended to other EvpC homologs for understanding the mechanism of T6SS and targeting T6SS mediated virulence in Gram-negative pathogens

Youth Savings Patterns and Performance in Colombia, Ghana, Kenya, and Nepal: YouthSave Research Report 2015

If offered an opportunity to save via formal financial services, will youth in developing countries participate, save, and accumulate assets? This is one of the key questions in YouthSave, a savings initiative implemented in four developing countries, targeting youth aged 12 to 18 years, from predominantly low-income households. This report presents two-year findings from a study that tracks account uptake and saving patterns and performance in youth savings accounts in four countries: Colombia, Ghana, Kenya, and Nepal. This savings demand assessment (SDA) is ambitious in its attempt to include systematic data on as many youth savers as possible. The result is a very large dataset that enables us to report in detail who is saving, and factors associated with saving patterns and performance. The report is divided into four sections: the ten key findings; the project summary; the body, which consists of Chapters 1 through 9 and summarizes information across all four countries; and the appendices, which include country-specific details and summary tables. A summary of findings appears at the end of each chapter

Youth Savings Patterns and Performance in Colombia, Ghana, Kenya, and Nepal

Youth Savings Patterns and Performance in Colombia, Ghana, Kenya, and Nepa

Youth Savings Patterns and Performance in Colombia, Ghana, Kenya, and Nepal: Executive Summary

This summary presents an overview of findings from the YouthSave Project\u27s 2015 research report Youth Savings Patterns and Performance in Columbia, Ghana, Kenya, and Nepal. Created in partnership with the MasterCard Foundation, YouthSave investigated the potential of savings accounts as a tool for youth development and financial inclusion in developing countries by co-designing tailored, sustainable savings products with local financial institutions and assessing their performance and development outcomes with local researchers. This study tracked account uptake, saving patterns, and savings performance in youth savings accounts in Colombia, Ghana, Kenya, and Nepal

Savings Patterns and Performance in Colombia, Ghana, Kenya, and Nepal

Savings Patterns and Performance in Colombia, Ghana, Kenya, and Nepa

Youth Savings Patterns and Performance in Colombia, Ghana, Kenya, and Nepal

Youth Savings Patterns and Performance in Colombia, Ghana, Kenya, and Nepa

Youth Saving Patterns and Performance in Colombia, Ghana, Kenya, and Nepal: Key Findings

If provided an opportunity to save via formal financial services, do youth in developing countries participate, save, and accumulate assets? This was one of the key questions asked in YouthSave. Savings accounts were created in four developing countries, targeting youth aged 12 to 18 years from predominantly low-income households. This brief highlights research findings on account uptake and savings from the Savings Demand Assessment (SDA)

Youth Savings Patterns and Performance in Colombia, Ghana, Kenya, and Nepal

Youth Savings Patterns and Performance in Colombia, Ghana, Kenya, and Nepa

Development of novel methods for non-canonical myeloma protein analysis with an innovative adaptation of immunofixation electrophoresis, native top-down mass spectrometry, and middle-down de novo sequencing

OBJECTIVES: Multiple myeloma (MM) is a malignant plasma cell neoplasm, requiring the integration of clinical examination, laboratory and radiological investigations for diagnosis. Detection and isotypic identification of the monoclonal protein(s) and measurement of other relevant biomarkers in serum and urine are pivotal analyses. However, occasionally this approach fails to characterize complex protein signatures. Here we describe the development and application of next generation mass spectrometry (MS) techniques, and a novel adaptation of immunofixation, to interrogate non-canonical monoclonal immunoproteins. METHODS: Immunoprecipitation immunofixation (IP-IFE) was performed on a Sebia Hydrasys Scan2. Middle-down de novo sequencing and native MS were performed with multiple instruments (21T FT-ICR, Q Exactive HF, Orbitrap Fusion Lumos, and Orbitrap Eclipse). Post-acquisition data analysis was performed using Xcalibur Qual Browser, ProSight Lite, and TDValidator. RESULTS: We adapted a novel variation of immunofixation electrophoresis (IFE) with an antibody-specific immunosubtraction step, providing insight into the clonal signature of gamma-zone monoclonal immunoglobulin (M-protein) species. We developed and applied advanced mass spectrometric techniques such as middle-down de novo sequencing to attain in-depth characterization of the primary sequence of an M-protein. Quaternary structures of M-proteins were elucidated by native MS, revealing a previously unprecedented non-covalently associated hetero-tetrameric immunoglobulin. CONCLUSIONS: Next generation proteomic solutions offer great potential for characterizing complex protein structures and may eventually replace current electrophoretic approaches for the identification and quantification of M-proteins. They can also contribute to greater understanding of MM pathogenesis, enabling classification of patients into new subtypes, improved risk stratification and the potential to inform decisions on future personalized treatment modalities