Co-development of vulnerability and risk assessment framework and methodology for Nepal.

Conducting a vulnerability and risk assessment (VRA) is a critical step in adaptation planning and implementation. This research paper has assessed how the co-developed approach helped the Government of Nepal and stakeholders in Nepal to agree on and operationalize the vulnerability and risk assessment framework and what works best for the country's context. The methodological framework consists of eight steps including (i) scoping, (ii) reviewing the framework (iii) identifying data sources (iv) exploring data sources, nature, and character (v) data collection, tabulation, filtration, and normalization (vi) weightage and composite value (vii) analysis of the data (viii) identifying climate change impact, vulnerability, and risk. The output of the vulnerability and risk assessment in Nepal presents a compilation of impacts, vulnerabilities, and risks and a description of their context, root causes, and trends. These research-based assessment can be used to draw some possible adaptation options and improved decision-making at the national and sub-national levels. However, there were some challenges faced in analyzing the vulnerability and risks based on the indicators. There was a lack of a multi-year, complete, and uniform database, and difficulties in developing scenarios of hazards due to unclarity on climate change attribution. The lessons from this paper will be important for designing a more practical and country-driven VRA framework and methodology for other countries.

Development and application of multicolor burst analysis spectroscopy.

The formation and disassembly of macromolecular particles is a ubiquitous and essential feature of virtually all living organisms. Additionally, diseases are often associated with the accumulation and propagation of biologically active nanoparticles, like the formation of toxic protein aggregates in protein misfolding diseases and the growth of infectious viral particles. The heterogeneous and dynamic nature of biologically active particles can make them exceedingly challenging to study. The single-particle fluorescence technique known as burst analysis spectroscopy (BAS) was developed to facilitate real-time measurement of macromolecular particle distributions in the submicron range in a minimally perturbing, free-solution environment. Here, we develop a multicolor version of BAS and employ it to examine two problems in macromolecular assembly: 1) the extent of DNA packing heterogeneity in bacteriophage viral particles and 2) growth models of non-native protein aggregates. We show that multicolor BAS provides a powerful and flexible approach to studying hidden properties of important biological particles like viruses and protein aggregates.

Clathrin coat disassembly by the yeast Hsc70/Ssa1p and auxilin/Swa2p proteins observed by single-particle burst analysis spectroscopy.

The role of clathrin-coated vesicles in receptor-mediated endocytosis is conserved among eukaryotes, and many of the proteins required for clathrin coat assembly and disassembly have orthologs in yeast and mammals. In yeast, dozens of proteins have been identified as regulators of the multistep reaction required for endocytosis, including those that regulate disassembly of the clathrin coat. In mammalian systems, clathrin coat disassembly has been reconstituted using neuronal clathrin baskets mixed with the purified chaperone ATPase 70-kDa heat shock cognate (Hsc70), plus a clathrin-specific co-chaperone, such as the synaptic protein auxilin. Yet, despite previous characterization of the yeast Hsc70 ortholog, Ssa1p, and the auxilin-like ortholog, Swa2p, testing mechanistic models for disassembly of nonneuronal clathrin coats has been limited by the absence of a functional reconstitution assay. Here we use single-particle burst analysis spectroscopy, in combination with fluorescence correlation spectroscopy, to follow the population dynamics of fluorescently tagged yeast clathrin baskets in the presence of purified Ssa1p and Swa2p. An advantage of this combined approach for mechanistic studies is the ability to measure, as a function of time, changes in the number and size of objects from a starting population to the reaction products. Our results indicate that Ssa1p and Swa2p cooperatively disassemble yeast clathrin baskets into fragments larger than the individual triskelia, suggesting that disassembly of clathrin-coated vesicles may proceed through a partially uncoated intermediate.