In eukaryotes, N-degron pathways (formerly “N-end rule pathways”) comprise a set of proteolytic systems whose unifying feature is their ability to recognize proteins containing N-terminal degradation signals called N-degrons, thereby causing degradation of these proteins by the 26S proteasome or autophagy. Gid4, a subunit of the GID ubiquitin ligase in the yeast Saccharomyces cerevisiae, is the recognition component (N-recognin) of the GID-mediated Pro/N-degron pathway. Gid4 targets proteins by recognizing their N-terminal Pro residues or a Pro at position 2, in the presence of distinct adjoining sequence motifs. Under conditions of low or absent glucose, cells make it through gluconeogenesis. When S. cerevisiae grows on a nonfermentable carbon source, its gluconeogenic enzymes Fbp1, Icl1, Mdh2, and Pck1 are expressed and long-lived. Transition to a medium containing glucose inhibits the synthesis of these enzymes and induces their degradation by the Gid4-dependent Pro/N-degron pathway. While studying yeast Gid4, we identified a similar but uncharacterized yeast protein (YGR066C), which we named Gid10. A screen for N-terminal peptide sequences that can bind to Gid10 showed that substrate specificities of Gid10 and Gid4 overlap but are not identical. Gid10 is not expressed under usual (unstressful) growth conditions, but is induced upon starvation or osmotic stresses. Using protein binding analyses and degradation assays with substrates of GID, we show that Gid10 can function as a specific N-recognin of the Pro/N-degron pathway.

Lateral-flow assays (LFAs) are rapid and inexpensive, yet they are nearly 1,000-fold less sensitive than laboratory-based tests. Here we show that plasmonically active antibody-conjugated fluorescent gold nanorods can make conventional LFAs ultrasensitive. With sample-to-answer times within 20 min, plasmonically enhanced LFAs read out via a standard benchtop fluorescence scanner attained about 30-fold improvements in dynamic range and in detection limits over 4-h-long gold-standard enzyme-linked immunosorbent assays, and achieved 95% clinical sensitivity and 100% specificity for antibodies in plasma and for antigens in nasopharyngeal swabs from individuals with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Comparable improvements in the assay's performance can also be achieved via an inexpensive portable scanner, as we show for the detection of interleukin-6 in human serum samples and of the nucleocapsid protein of SARS-CoV-2 in nasopharyngeal samples. Plasmonically enhanced LFAs outperform standard laboratory tests in sensitivity, speed, dynamic range, ease of use and cost, and may provide advantages in point-of-care diagnostics.Lateral-flow assays (LFAs) are among the simplest, fastest and cheapest point-of-care (POC) diagnostic methods, and offer broad potential for population-level screening for disease 1,2 . Although numerous LFAs for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies 3-5 and antigens 6,7 have been introduced, none has sensitivity and quantitation comparable to laboratory-based diagnostics such as real-time PCR with reverse transcription (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) [8][9][10] . In general, conventional colorimetric LFAs are ~1,000-fold less sensitive than these standard laboratory tests 11,12 , and diagnosis using LFAs requires an additional confirmatory laboratory-based test to correctly establish negative results. Colorimetric LFAs are often inadequate for quantitative read-outs, owing to limited changes in colour with respect to the variation of the concentration of the target analyte 13 .

Analysis of high order correlations in fluorescence fluctuation spectroscopy was developed in the late 1980s but since then has been replaced by alternative brightness analysis methods. However, high order correlation has important advantages in many experiments. We present a new cumulantbased formalism of high order correlation that greatly simplifies data analysis. The new formalism is used to derive general expressions for variance of high order correlations that show good agreement with experiment in a model system of fluorescently-labeled DNA oligomers. A simulation of binary systems in which both diffusion time and brightness are varied illustrates clearly that high order analysis has better sensitivity to brightness than fluorescence correlation spectroscopy (FCS). These results have implications for analysis of isomerization reactions and dual-beam FCS with flow. We also demonstrate that high order correlations can detect photobleaching in the observation volume. The application of this formalism to many FCS-based experiments allows more accurate analysis in addition to describing more molecular parameters.

Eukaryotic N-degron pathways are proteolytic systems whose unifying feature is their ability to recognize proteins containing N-terminal (Nt) degradation signals called N-degrons, and to target these proteins for degradation by the 26S proteasome or autophagy. GID4, a subunit of the GID ubiquitin ligase, is the main recognition component of the proline (Pro)/N-degron pathway. GID4 targets proteins through their Nt-Pro residue or a Pro at position 2, in the presence of specific downstream sequence motifs. Here we show that human GID4 can also recognize hydrophobic Nt-residues other than Pro. One example is the sequence Nt-IGLW, bearing Nt-Ile. Nt-IGLW binds to wild-type human GID4 with aKdof 16 μM, whereas the otherwise identical Nt-Pro–bearing sequence PGLW binds to GID4 more tightly, with aKdof 1.9 μM. Despite this difference in affinities of GID4 for Nt-IGLW vs. Nt-PGLW, we found that the GID4-mediated Pro/N-degron pathway of the yeastSaccharomyces cerevisiaecan target an Nt-IGLW–bearing protein for rapid degradation. We solved crystal structures of human GID4 bound to a peptide bearing Nt-Ile or Nt-Val. We also altered specific residues of human GID4 and measured the affinities of resulting mutant GID4s for Nt-IGLW and Nt-PGLW, thereby determining relative contributions of specific GID4 residues to the GID4-mediated recognition of Nt-Pro vs. Nt-residues other than Pro. These and related results advance the understanding of targeting by the Pro/N-degron pathway and greatly expand the substrate recognition range of the GID ubiquitin ligase in both human and yeast cells.

Simple, simpler, simplest: Spontaneous pattern formation in a commonplace system Am. J. Phys. 80, 578 (2012) Determination of contact angle from the maximum height of enlarged drops on solid surfaces Am. J. Phys. 80, 284 (2012) Aerodynamics in the classroom and at the ball park Am. J. Phys. 80, 289 (2012) The added mass of a spherical projectile Am.An effective one-mass model of phonation is developed. It borrows the salient features of the classic two-mass model of human speech developed by Ishizaka, Matsudaira, and Flanagan. Their model is based on the idea that the oscillating vocal folds maintain their motion by deriving energy from the flow of air through the glottis. We argue that the essence of the action of the aerodynamic forces on the vocal folds is captured by negative Coulomb damping, which acts on the oscillator to energize it. A viscous force is added to include the effects of tissue damping. The solutions to this single oscillator model show that when it is excited by negative Coulomb damping, it will reach a limit cycle. Displacements, phase portraits, and energy histories are presented for two underdamped linear oscillators. A nonlinear force is added so that the variations of the fundamental frequency and the open quotient with lung pressure are comparable to the behavior of the two-mass model.