Nanoaperture fabrication via colloidal lithography for single molecule fluorescence analysis

In single molecule fluorescence studies, background emission from labeled substrates often restricts their concentrations to non-physiological nanomolar values. One approach to address this challenge is the use of zero-mode waveguides (ZMWs), nanoscale holes in a thin metal film that physically and optically confine the observation volume allowing much higher concentrations of fluorescent substrates. Standard fabrication of ZMWs utilizes slow and costly E-beam nano-lithography. Herein, ZMWs are made using a self-assembled mask of polystyrene microspheres, enabling fabrication of thousands of ZMWs in parallel without sophisticated equipment. Polystyrene 1 mu m dia. microbeads self-assemble on a glass slide into a hexagonal array, forming a mask for the deposition of metallic posts in the inter-bead interstices. The width of those interstices (and subsequent posts) is adjusted within 100-300 nm by partially fusing the beads at the polystyrene glass transition temperature. The beads are dissolved in toluene, aluminum or gold cladding is deposited around the posts, and those are dissolved, leaving behind an array ZMWs. Parameter optimization and the performance of the ZMWs are presented. By using colloidal self-assembly, typical laboratories can make use of sub-wavelength ZMW technology avoiding the availability and expense of sophisticated clean-room environments and equipment

Mediterranean marine protected areas have higher biodiversity via increased evenness, not abundance

1. Protected areas are central to biodiversity conservation. For marine fish, marine protected areas (MPAs) often harbour more individuals, especially of species targeted by fisheries. But precise pathways of biodiversity change remain unclear. For example, how local-scale responses combine to affect regional biodiversity, important for managing spatial networks of MPAs, is not well known. Protection potentially influences three components of fish assemblages that determine how species accumulate with sampling effort and spatial scale: the total number of individuals, the relative abundance of species and within-species aggregation. Here, we examined the contributions of each component to species richness changes inside MPAs as a function of spatial scale. 2. Using standardized underwater visual survey data, we measured the abundance and species richness of reef fishes in 43 protected and 41 fished sites in the Mediterranean Sea. 3. At both local and regional scales, increased species evenness caused by added common species in MPAs compared to fished sites was the most important proximate driver of higher diversity. 4. Site-to-site variation in the composition (i.e. β-diversity) of common species was also higher among protected sites, and depended on sensitivity to exploitation. There were more abundant exploited species at regional scales than at local scales, reflecting a tendency for different protected sites to harbour different exploited species. In contrast, fewer abundant unexploited species were found at the regional scale than at the local scale, meaning that relative abundances at the regional scale were less even than at the local scale. 5. Synthesis and applications. Although marine protected areas (MPAs) are known to strongly influence fish community abundance and biomass, we found that changes to the relative abundance of species (i.e. increased evenness) dominated the biodiversity response to protection. MPAs had more relatively common species, which in turn led to higher diversity for a given sampling effort. Moreover, higher β-diversity of common species meant that local-scale responses were magnified at the regional scale due to site-to-site variation inside protected areas for exploited species. Regional conservation efforts can be strengthened by examining how multiple components of biodiversity respond to protection across spatial scales

Modeling spatiotemporally varying protein–protein interactions in CyLaKS, the Cytoskeleton Lattice-based Kinetic Simulator

Interaction of cytoskeletal filaments, motor proteins, and crosslinking proteins drives important cellular processes such as cell division and cell movement. Cytoskeletal networks also exhibit nonequilibrium self-assembly in reconstituted systems. An emerging problem in cytoskeletal modeling and simulation is spatiotemporal alteration of the dynamics of filaments, motors, and associated proteins. This can occur due to motor crowding, obstacles along the filament, motor interactions and direction switching, and changes, defects, or heterogeneity in the filament binding lattice. How such spatiotemporally varying cytoskeletal filaments and motor interactions affect their collective properties is not fully understood. We developed the Cytoskeleton Lattice-based Kinetic Simulator (CyLaKS) to investigate such problems. The simulation model builds on previous work by incorporating motor mechanochemistry into a simulation with many interacting motors and/or associated proteins on a discretized lattice. CyLaKS also includes detailed balance in binding kinetics, movement, and lattice heterogeneity. The simulation framework is flexible and extensible for future modeling work and is available on GitHub for others to freely use or build upon. Here we illustrate the use of CyLaKS to study long-range motor interactions, microtubule lattice heterogeneity, motion of a heterodimeric motor, and how changing crosslinker number affects filament separation

Nanoaperture fabrication via colloidal lithography for single molecule fluorescence analysis.

In single molecule fluorescence studies, background emission from labeled substrates often restricts their concentrations to non-physiological nanomolar values. One approach to address this challenge is the use of zero-mode waveguides (ZMWs), nanoscale holes in a thin metal film that physically and optically confine the observation volume allowing much higher concentrations of fluorescent substrates. Standard fabrication of ZMWs utilizes slow and costly E-beam nano-lithography. Herein, ZMWs are made using a self-assembled mask of polystyrene microspheres, enabling fabrication of thousands of ZMWs in parallel without sophisticated equipment. Polystyrene 1 μm dia. microbeads self-assemble on a glass slide into a hexagonal array, forming a mask for the deposition of metallic posts in the inter-bead interstices. The width of those interstices (and subsequent posts) is adjusted within 100-300 nm by partially fusing the beads at the polystyrene glass transition temperature. The beads are dissolved in toluene, aluminum or gold cladding is deposited around the posts, and those are dissolved, leaving behind an array ZMWs. Parameter optimization and the performance of the ZMWs are presented. By using colloidal self-assembly, typical laboratories can make use of sub-wavelength ZMW technology avoiding the availability and expense of sophisticated clean-room environments and equipment

Same data, different analysts: variation in effect sizes due to analytical decisions in ecology and evolutionary biology

Gould E, Fraser H, Parker T, et al. Same data, different analysts: variation in effect sizes due to analytical decisions in ecology and evolutionary biology. 2023.Although variation in effect sizes and predicted values among studies of similar phenomena is inevitable, such variation far exceeds what might be produced by sampling error alone. One possible explanation for variation among results is differences among researchers in the decisions they make regarding statistical analyses. A growing array of studies has explored this analytical variability in different (mostly social science) fields, and has found substantial variability among results, despite analysts having the same data and research question. We implemented an analogous study in ecology and evolutionary biology, fields in which there have been no empirical exploration of the variation in effect sizes or model predictions generated by the analytical decisions of different researchers. We used two unpublished datasets, one from evolutionary ecology (blue tit, Cyanistes caeruleus, to compare sibling number and nestling growth) and one from conservation ecology (Eucalyptus, to compare grass cover and tree seedling recruitment), and the project leaders recruited 174 analyst teams, comprising 246 analysts, to investigate the answers to prespecified research questions. Analyses conducted by these teams yielded 141 usable effects for the blue tit dataset, and 85 usable effects for the Eucalyptus dataset. We found substantial heterogeneity among results for both datasets, although the patterns of variation differed between them. For the blue tit analyses, the average effect was convincingly negative, with less growth for nestlings living with more siblings, but there was near continuous variation in effect size from large negative effects to effects near zero, and even effects crossing the traditional threshold of statistical significance in the opposite direction. In contrast, the average relationship between grass cover and Eucalyptus seedling number was only slightly negative and not convincingly different from zero, and most effects ranged from weakly negative to weakly positive, with about a third of effects crossing the traditional threshold of significance in one direction or the other. However, there were also several striking outliers in the Eucalyptus dataset, with effects far from zero. For both datasets, we found substantial variation in the variable selection and random effects structures among analyses, as well as in the ratings of the analytical methods by peer reviewers, but we found no strong relationship between any of these and deviation from the meta-analytic mean. In other words, analyses with results that were far from the mean were no more or less likely to have dissimilar variable sets, use random effects in their models, or receive poor peer reviews than those analyses that found results that were close to the mean. The existence of substantial variability among analysis outcomes raises important questions about how ecologists and evolutionary biologists should interpret published results, and how they should conduct analyses in the future