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Cellular mechanotransduction, a process central to cell biology, embryogenesis, adult physiology, and multiple diseases, is thought to be mediated by force-driven changes in protein conformation that control protein function. However, methods to study proteins under defined mechanical loads on a biochemical scale are lacking. We report the development of a DNA-based device in which the transition between single- and double-stranded DNA applies tension to an attached protein. Using a fragment of the talin rod domain as a test case, negative-stain electron microscopy reveals programmable extension, while pull down assays show tension-induced binding to two ligands, ARPC5L and vinculin, known to bind to cryptic sites inside the talin structure. These results demonstrate the utility of the DNA clamp for biochemical studies and potential structural analysis.

Fluorescence microscopy has been one of the most discovery-rich methods in biology. In the digital age, the discipline is becoming increasingly quantitative. Virtually all biological laboratories have access to fluorescence microscopes, but abilities to quantify biomolecule copy numbers are limited by the complexity and sophistication associated with current quantification methods. Here, we present DNA-origami-based fluorescence brightness standards for counting 5-300 copies of proteins in bacterial and mammalian cells, tagged with fluorescent proteins or membrane-permeable organic dyes. Compared to conventional quantification techniques, our brightness standards are robust, straightforward to use, and compatible with nearly all fluorescence imaging applications, thereby providing a practical and versatile tool to quantify biomolecules via fluorescence microscopy.

Waste generation is expected to increase in most countries for many decades with landfill disposal still the dominant solid waste management method 1-3 . Yet, operational characteristics of landfills are often poorly understood with comparative statistics substantially lacking. Here, we call for a more formal waste informatics to organize and standardize waste management knowledge at multiple spatial scales through analysis of recently reported data from 1232 U.S. landfills and other high resolution data sets. We create the first known estimate of available U.S. municipal waste stocks (8.5 billion tonnes) and go on to resolve these stocks at the county level, reflecting prospective urban mining opportunities. Our analysis of disposal rates and landfill capacities reveals that more than half of U.S. states have more than 25 years of life remaining. We also estimate the gross energy potential of landfill gas in the U.S. (338 billion MJ/yr) by examining 922 operational methane collection systems and demonstrate that the greatest energy recovery opportunities lie at landfills with existing collection systems and energy conversion infrastructure. Finally, we found that the number of landfills reaching the federally defined 30-year postclosure care period will more than triple in the coming two decades, with 264 sites expected by the year 2044, highlighting the need to develop and standardize metrics carefully to define and standardize when it is appropriate to end or scale back long-term landfill monitoring.

More than 1,500 construction and demolition debris (CDD) landfills operate in the United States (U.S.), and U.S. federal regulations do not require containment features such as low-permeability liners and leachate collection systems for these facilities. Here we evaluate groundwater quality from samples collected in groundwater monitoring networks at 91 unlined, permitted CDD landfills in Florida, U.S. A total of 460,504 groundwater sample results were analyzed, with a median of 10 years of quarterly or semiannual monitoring data per site including more than 400 different chemical constituents. Downgradient concentrations of total dissolved solids, sulfate, chloride, iron, ammonia-nitrogen, and aluminum were greater than upgradient concentrations (p < 0.05). At downgradient wells where sulfate concentrations were greater than 150 mg/L (approximately 10% of the maximum dissolved sulfate concentration in water, which suggests the presence of leachate from the landfill), iron and arsenic were detected in 91% and 43% of samples, with median concentrations of 1,900 μg/L and 11 μg/L, respectively. These results show that although health-based standards can be exceeded at unlined CDD landfills, the magnitude of detected chemical concentrations is generally small and reflective of leached minerals from components (wood, concrete, and gypsum drywall) that comprise the bulk of discarded CDD by mass.

In the social amoeba Dictyostelium, Skp1 is hydroxylated on proline 143 and further modified by three cytosolic glycosyltransferases to yield an O-linked pentasaccharide that contributes to O2 regulation of development. Skp1 is an adapter in the Skp1/cullin1/F-box protein family of E3 ubiquitin ligases that targets specific proteins for polyubiquitination and subsequent proteasomal degradation. To investigate the biochemical consequences of glycosylation, untagged full-length Skp1 and several of its posttranslationally modified isoforms were expressed and purified to near homogeneity using recombinant and in vitro strategies. Interaction studies with the soluble mammalian F-box protein Fbs1/Fbg1/OCP1 revealed preferential binding to the glycosylated isoforms of Skp1. This difference correlated with the increased α-helical and decreased β-sheet content of glycosylated Skp1s based on circular dichroism and increased folding order based on small-angle X-ray scattering. A comparison of the molecular envelopes of fully glycosylated Skp1 and the apoprotein indicated that both isoforms exist as an antiparallel dimer that is more compact and extended in the glycosylated state. Analytical gel filtration and chemical cross-linking studies showed a growing tendency of less modified isoforms to dimerize. Considering that regions of free Skp1 are intrinsically disordered and Skp1 can adopt distinct folds when bound to F-box proteins, we propose that glycosylation, which occurs adjacent to the F-box binding site, influences the spectrum of energetically similar conformations that vary inversely in their propensity to dock with Fbs1 or another Skp1. Glycosylation may thus influence Skp1 function by modulating F-box protein binding in cells.

Recent research and policy directives have emerged with a focus on sustainable management of waste materials, and the mining of old landfills represents an opportunity to meet sustainability goals by reducing the release of liquid- and gas-phase contaminants into the environment, recovering land for more productive use, and recovering energy from the landfilled materials. The emissions associated with the landfill mining process (waste excavation, screening, and on-site transportation) were inventoried on the basis of diesel fuel consumption data from two full-scale mining projects (1.3-1.5 L/in-place m(3) of landfill space mined) and unit emissions (mass per liter of diesel consumption) from heavy equipment typically deployed for mining landfills. An analytical framework was developed and used in an assessment of the life-cycle environmental impacts of a few end-use management options for materials deposited and mined from an unlined landfill. The results showed that substantial greenhouse gas emission reductions can be realized in both the waste relocation and materials and energy recovery scenarios compared to a "do nothing" case. The recovery of metal components from landfilled waste was found to have the greatest benefit across nearly all impact categories evaluated, while emissions associated with heavy equipment to mine the waste itself were found to be negligible compared to the benefits that mining provided.

The dialdehyde produced by the periodate cleavage of the ribose moiety of uridine 5'-diphosphate (UDP) has been used as an affinity label for the UDP-galactose/UDP binding site of galactosyltransferase from bovine colostrum. This derivative causes progressive inactivation of galactosyltransferase at a rate dependent on its concentration, and under certain conditions is a competitive inhibitor with respect to UDP-galactose. The substrate UDP-galactose protects the enzyme from inactivation. The inactivation is also dependent on Mn2+ concentration in a range that implies that the binding of Mn2+ at site I is a prerequisite for the binding of the UDP derivative. The inactivation can be progressively reversed by nitrogenous bases, or stabilized by KBH4 reduction, which is consistent with the hypothesis that a Schiff base has formed with a lysine residue. Galactosyltransferase was inactivated with a [3H]UDP derivative and the predominant labeled peptide, from thermolysin digestion, isolated and characterized as: Ser-Gly-Lys-UDP.