Quantitative comparison of nuclear transport inhibition by SARS coronavirus ORF6 reveals the importance of oligomerization.

Open Reading Frame 6 (ORF6) proteins, which are unique to severe acute respiratory syndrome-related (SARS) coronavirus, inhibit the classical nuclear import pathway to antagonize host antiviral responses. Several alternative models were proposed to explain the inhibitory function of ORF6 [H. Xia .

Multiplexed single-cell analysis of FNA allows accurate diagnosis of salivary gland tumors.

Diagnosing salivary gland tumors (SGTs) through fine-needle aspiration (FNA) biopsies is challenging due to the overlapping cytomorphologic features between benign and malignant tumors. The authors developed an innovative, multiplexed cycling technology for the rapid analyses of single cells obtained from FNA that can facilitate the molecular analyses and diagnosis of SGTs. Antibodies against 29 protein markers associated with 7 SGT subtypes were validated and chemically modified via custom linker-bio-orthogonal probes (FAST).

High-Content Screening and Computational Prediction Reveal Viral Genes That Suppress the Innate Immune Response.

Suppression of the host innate immune response is a critical aspect of viral replication. Upon infection, viruses may introduce one or more proteins that inhibit key immune pathways, such as the type I interferon pathway. However, the ability to predict and evaluate viral protein bioactivity on targeted pathways remains challenging and is typically done on a single-virus or -gene basis.

Quantification of nuclear transport inhibition by SARS-CoV-2 ORF6 using a broadly applicable live-cell dose-response pipeline.

SARS coronavirus ORF6 inhibits the classical nuclear import pathway to antagonize host antiviral responses. Several models were proposed to explain its inhibitory function, but quantitative measurement is needed for model evaluation and refinement. We report a broadly applicable live-cell method for calibrated dose-response characterization of the nuclear transport alteration by a protein of interest.

O-GlcNAc modification of nuclear pore complexes accelerates bidirectional transport.

Macromolecular transport across the nuclear envelope depends on facilitated diffusion through nuclear pore complexes (NPCs). The interior of NPCs contains a permeability barrier made of phenylalanine-glycine (FG) repeat domains that selectively facilitates the permeation of cargoes bound to nuclear transport receptors (NTRs). FG-repeat domains in NPCs are a major site of O-linked N-acetylglucosamine (O-GlcNAc) modification, but the functional role of this modification in nucleocytoplasmic transport is unclear.

Label-free assessment of pre-implantation embryo quality by the Fluorescence Lifetime Imaging Microscopy (FLIM)-phasor approach.

Development of quantitative, safe and rapid techniques for assessing embryo quality provides significant advances in Assisted Reproductive Technologies (ART). Instead of assessing the embryo quality by the standard morphologic evaluation, we apply the phasor-FLIM (Fluorescence Lifetime Imaging Microscopy) method to capture endogenous fluorescent biomarkers of pre-implantation embryos as a non-morphological caliber for embryo quality. Here, we identify, under hypoxic and non-hypoxic conditions, the unique spectroscopic trajectories at different stages of mouse pre-implantation development, which is referred to as the developmental, or "D-trajectory", that consists of fluorescence lifetime from different stages of mouse pre-implantation embryos.

Central-spindle microtubules are strongly coupled to chromosomes during both anaphase A and anaphase B.

Spindle microtubules, whose dynamics vary over time and at different locations, cooperatively drive chromosome segregation. Measurements of microtubule dynamics and spindle ultrastructure can provide insight into the behaviors of microtubules, helping elucidate the mechanism of chromosome segregation. Much work has focused on the dynamics and organization of kinetochore microtubules, that is, on the region between chromosomes and poles.

Measuring NDC80 binding reveals the molecular basis of tension-dependent kinetochore-microtubule attachments.

Proper kinetochore-microtubule attachments, mediated by the NDC80 complex, are required for error-free chromosome segregation. Erroneous attachments are corrected by the tension dependence of kinetochore-microtubule interactions. Here, we present a method, based on fluorescence lifetime imaging microscopy and Förster resonance energy transfer, to quantitatively measure the fraction of NDC80 complexes bound to microtubules at individual kinetochores in living human cells.

Chromosomal passenger complex hydrodynamics suggests chaperoning of the inactive state by nucleoplasmin/nucleophosmin.

The chromosomal passenger complex (CPC) is a conserved, essential regulator of cell division. As such, significant anti-cancer drug development efforts have been focused on targeting it, most notably by inhibiting its AURKB kinase subunit. The CPC is activated by AURKB-catalyzed autophosphorylation on multiple subunits, but how this regulates CPC interactions with other mitotic proteins remains unclear.

Bridging length scales to measure polymer assembly.

Time-resolvable quantitative measurements of polymer concentration are very useful to elucidate protein polymerization pathways. There are numerous techniques to measure polymer concentrations in purified protein solutions, but few are applicable in vivo. Here we develop a methodology combining microscopy and spectroscopy to overcome the limitations of both approaches for measuring polymer concentration in cells and cell extracts.

Developing and Testing a Bayesian Analysis of Fluorescence Lifetime Measurements.

FRET measurements can provide dynamic spatial information on length scales smaller than the diffraction limit of light. Several methods exist to measure FRET between fluorophores, including Fluorescence Lifetime Imaging Microscopy (FLIM), which relies on the reduction of fluorescence lifetime when a fluorophore is undergoing FRET. FLIM measurements take the form of histograms of photon arrival times, containing contributions from a mixed population of fluorophores both undergoing and not undergoing FRET, with the measured distribution being a mixture of exponentials of different lifetimes.

Identification of a Novel De Novo Mutation of the TAZ Gene in a Korean Patient with Barth Syndrome.

Barth syndrome (BTHS) is a rare genetic disorder characterized by various types of cardiomyopathy, neutropenia, failure to thrive, skeletal myopathy, and 3-methylglutaconic aciduria. BTHS is caused by loss-of-function mutations in the tafazzin (TAZ) gene located on chromosome Xq28, leading to cardiolipin deficiency. We report a 13-month-old boy with BTHS who had a novel de novo mutation in the TAZ gene.

Studying Kinetochores In Vivo Using FLIM-FRET.

Kinetochores play essential roles in coordinating mitosis, as a mechanical connector between chromosome and microtubule and as a source of numerous biochemical signals. These mechanical and biochemical behaviors of kinetochores change dynamically in cells during mitosis. Therefore, understanding kinetochore function requires an imaging tool that quantifies the protein-protein interactions or biochemical changes with high spatiotemporal resolution.

Measuring microtubule polarity in spindles with second-harmonic generation.

The spatial organization of microtubule polarity, and the interplay between microtubule polarity and protein localization, is thought to be crucial for spindle assembly, anaphase, and cytokinesis, but these phenomena remain poorly understood, in part due to the difficulty of measuring microtubule polarity in spindles. We develop and implement a method to nonperturbatively and quantitatively measure microtubule polarity throughout spindles using a combination of second-harmonic generation and two-photon fluorescence. We validate this method using computer simulations and by comparison to structural data on spindles obtained from electron tomography and laser ablation.

The folding transition state of protein L is extensive with nonnative interactions (and not small and polarized).

Progress in understanding protein folding relies heavily upon an interplay between experiment and theory. In particular, readily interpretable experimental data that can be meaningfully compared to simulations are required. According to standard mutational ϕ analysis, the transition state for Protein L contains only a single hairpin.

Small-angle X-ray scattering and single-molecule FRET spectroscopy produce highly divergent views of the low-denaturant unfolded state.

The results of more than a dozen single-molecule Förster resonance energy transfer (smFRET) experiments suggest that chemically unfolded polypeptides invariably collapse from an expanded random coil to more compact dimensions as the denaturant concentration is reduced. In sharp contrast, small-angle X-ray scattering (SAXS) studies suggest that, at least for single-domain proteins at non-zero denaturant concentrations, such compaction may be rare. Here, we explore this discrepancy by studying protein L, a protein previously studied by SAXS (at 5 °C), which suggested fixed unfolded-state dimensions from 1.

Allergic contact dermatitis from hair dye and development of lichen simplex chronicus.

Those who dye their hair frequently manifest allergic contact dermatitis (ACD) from p-phenylenediamine (PPD)-containing hair dye. PPD is known to be the most frequent sensitizer in hair dye, but there has been no documentation of this sensitizer having a role in chronic dermatologic conditions. Our department experienced a case of a 62-year-old woman with lichen simplex chronicus (LSC), who complained of aggravation after hair dyeing and made such an association.