The prognostic role of functional dependency in older inpatients with COVID-19.

BACKGROUND: Coronavirus disease 2019 (COVID-19) is a pandemic infection with substantial risk of death, especially in elderly persons. Information about the prognostic significance of functional status in older patients with COVID-19 is scarce. METHODS: Demographic, clinical, laboratory and short-term mortality data were collected of 186 consecutive patients aged ≥ 65 years hospitalized with COVID-19. The data were compared between 4 study groups: (1) age 65-79 years without severe functional dependency; (2) age ≥ 80 years without severe functional dependency; (3) age 65-79 years with severe functional dependency; and (4) age ≥ 80 years with severe functional dependency. Multivariate logistic regressions were performed to evaluate the variables that were most significantly associated with mortality in the entire sample. RESULTS: Statistically significant differences were observed between the groups in the proportions of males (p = 0.007); of patients with diabetes mellitus (p = 0.025), cerebrovascular disease (p < 0.001), renal failure (p = 0.003), dementia (p < 0.001), heart failure (p = 0.005), pressure sores (p < 0.001) and malignant disorders (p = 0.007); and of patients residing in nursing homes (p < 0.001). Compared to groups 1 (n = 69) and 2 (n = 28), patients in groups 3 (n = 32) and 4 (n = 57) presented with lower mean serum albumin levels on admission (p < 0.001), and were less often treated with convalescent plasma (p < 0.001), tocilizumab (p < 0.001) and remdesivir (p < 0.001). The overall mortality rate was 23.1 %. The mortality rate was higher in group 4 than in groups 1 - 3: 45.6 % vs. 8.7 %, 17.9% and 18.3 %, respectively (p < 0.001). On multivariate analysis, both age ≥ 80 years and severe functional dependency were among the variables most significantly associated with mortality in the entire cohort (odds ratio [OR] 4.83, 95 % confidence interval [CI] 1.88 - 12.40, p < 0.001 and OR 2.51, 95 % CI 1.02 - 6.15, p = 0.044, respectively). Age ≥ 80 years with severe functional dependency (group 4) remained one of the variables most significantly associated with mortality (OR 10.42, 95 % CI 3.27-33.24 and p < 0.001). CONCLUSIONS: Among patients with COVID-19, the association of severe functional dependency with mortality is stronger among those aged ≥ 80 years than aged 65-79 years. Assessment of functional status may contribute to decision making for care of older inpatients with COVID-19.

Spatial sorting enables comprehensive characterization of liver zonation.

The mammalian liver is composed of repeating hexagonal units termed lobules. Spatially resolved single-cell transcriptomics revealed that about half of hepatocyte genes are differentially expressed across the lobule, yet technical limitations impeded reconstructing similar global spatial maps of other hepatocyte features. Here, we show how zonated surface markers can be used to sort hepatocytes from defined lobule zones with high spatial resolution. We apply transcriptomics, miRNA array measurements and mass spectrometry proteomics to reconstruct spatial atlases of multiple zonated features. We demonstrate that protein zonation largely overlaps with mRNA zonation, with the periportal HNF4α as an exception. We identify zonation of miRNAs such as miR-122, and inverse zonation of miRNAs and their hepatocyte target genes, highlighting potential regulation of protein levels through zonated mRNA degradation. Among the targets we find the pericentral Wnt receptors Fzd7 and Fzd8 and the periportal Wnt inhibitors Tcf7l1 and Ctnnbip1. Our approach facilitates reconstructing spatial atlases of multiple cellular features in the liver and other structured tissues.

Rapid labelling and covalent inhibition of intracellular native proteins using ligand-directed N-acyl-N-alkyl sulfonamide.

Selective modification of native proteins in live cells is one of the central challenges in recent chemical biology. As a unique bioorthogonal approach, ligand-directed chemistry recently emerged, but the slow kinetics limits its scope. Here we successfully overcome this obstacle using N-acyl-N-alkyl sulfonamide as a reactive group. Quantitative kinetic analyses reveal that ligand-directed N-acyl-N-alkyl sulfonamide chemistry allows for rapid modification of a lysine residue proximal to the ligand binding site of a target protein, with a rate constant of ~104 M-1 s-1, comparable to the fastest bioorthogonal chemistry. Despite some off-target reactions, this method can selectively label both intracellular and membrane-bound endogenous proteins. Moreover, the unique reactivity of N-acyl-N-alkyl sulfonamide enables the rational design of a lysine-targeted covalent inhibitor that shows durable suppression of the activity of Hsp90 in cancer cells. This work provides possibilities to extend the covalent inhibition approach that is currently being reassessed in drug discovery.

Method for Labeling Transcripts in Individual Escherichia coli Cells for Single-molecule Fluorescence In Situ Hybridization Experiments.

A method is described for labeling individual messenger RNA (mRNA) transcripts in fixed bacteria for use in single-molecule fluorescence in situ hybridization (smFISH) experiments in E. coli. smFISH allows the measurement of cell-to-cell variability in mRNA copy number of genes of interest, as well as the subcellular location of the transcripts. The main steps involved are fixation of the bacterial cell culture, permeabilization of cell membranes, and hybridization of the target transcripts with sets of commercially available short fluorescently-labeled oligonucleotide probes. smFISH can allow the imaging of the transcripts of multiple genes in the same cell, with limitations imposed by the spectral overlap between different fluorescent markers. Following completion of the protocol illustrated below, cells can be readily imaged using a microscope coupled with a camera suitable for low-intensity fluorescence. These images, together with cell contours obtained from segmentation of phase contrast frames, or from cell membrane staining, allow the calculation of the mRNA copy number distribution of a sample of cells using open-source or custom-written software. The labeling method described here can also be applied to image transcripts with stochastic optical reconstruction microscopy (STORM).