Ultrasensitive lateral-flow assays via plasmonically active antibody-conjugated fluorescent nanoparticles.

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).

Fluorescence Correlation Spectroscopy and Photon Counting Histograms in Finite, Bounded Domains.

Analysis of fluctuations arising as fluorescent particles pass through a focused laser beam has enabled quantitative characterization of a broad range of molecular kinetic processes. Two key mathematical frameworks that have enabled these quantifications are fluorescence correlation spectroscopy (FCS) and photon counting histogram (PCH) analysis. Although these frameworks are effective and accurate when the focused laser beam is well approximated by an infinite Gaussian beam with a waist that is small compared to the size of the region over which the fluorescent particles can diffuse, they cannot be applied to situations in which this region is bounded at the nanoscale.

Recognition of nonproline N-terminal residues by the Pro/N-degron pathway.

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.

Evolution of Substrates and Components of the Pro/N-Degron Pathway.

Gid4, a subunit of the ubiquitin ligase GID, is the recognition component of the Pro/N-degron pathway. Gid4 targets proteins in particular through their N-terminal (Nt) proline (Pro) residue. In and other yeasts, the gluconeogenic enzymes Fbp1, Icl1, and Mdh2 bear Nt-Pro and are conditionally destroyed by the Pro/N-degron pathway.

Gid10 as an alternative N-recognin of the Pro/N-degron pathway.

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 , 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.

Investigation of Nanoscopic Phase Separations in Lipid Membranes Using Inverse FCS.

Measurement of the sizes of nanoscopic particles is a difficult challenge, especially in two-dimensional systems such as cell membranes. We have extended inverse fluorescence correlation spectroscopy (iFCS) to endow it with unique advantages for measuring particle size from the nano- to the microscale. We have augmented iFCS with an analysis of moments of fluorescence fluctuations and used it to measure stages of phase separation in model lipid bilayer membranes.

New mechanisms that regulate Saccharomyces cerevisiae short peptide transporter achieve balanced intracellular amino acid concentrations.

The budding yeast Saccharomyces cerevisiae is able to take up large quantities of amino acids in the form of di- and tripeptides via a short peptide transporter, Ptr2p. It is known that PTR2 can be induced by certain peptides and amino acids, and the mechanisms governing this upregulation are understood at the molecular level. We describe two new opposing mechanisms of regulation that emphasize potential toxicity of amino acids: the first is upregulation of PTR2 in a population of cells, caused by amino acid secretion that accompanies peptide uptake; the second is loss of Ptr2p activity, due to transporter internalization following peptide uptake.

Effect of loop composition on the stability and folding kinetics of RNA hairpins with large loops.

RNA hairpins are ubiquitous structural elements in biological RNAs, where they have the potential to regulate RNA folding and interactions with other molecules. There are established methods for predicting the thermodynamic stability of an RNA hairpin, but there are still relatively few detailed examinations of the kinetics of folding. Nonetheless, several recent studies indicate that hairpin folding does not proceed via a simple two-state model.

Confidence intervals for concentration and brightness from fluorescence fluctuation measurements.

The theory of photon count histogram (PCH) analysis describes the distribution of fluorescence fluctuation amplitudes due to populations of fluorophores diffusing through a focused laser beam and provides a rigorous framework through which the brightnesses and concentrations of the fluorophores can be determined. In practice, however, the brightnesses and concentrations of only a few components can be identified. Brightnesses and concentrations are determined by a nonlinear least-squares fit of a theoretical model to the experimental PCH derived from a record of fluorescence intensity fluctuations.

A natural missing link between activated and downhill protein folding scenarios.

We propose protein PTB1 : 4W as a good candidate for engineering into a downhill folder. PTB1 : 4W has a probe-dependent thermal unfolding curve and sub-millisecond T-jump relaxation kinetics on more than one time scale. Its refolding rate in denaturant is a non-linear function of denaturant concentration (curved chevron plot).

Revival of high-order fluorescence correlation analysis: generalized theory and biochemical applications.

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 cumulant-based formalism of high-order correlation that greatly simplifies data analysis.