The new Coronavirus SARS-CoV-2 has infected more than three million people worldwide so far. Brazil is currently the second leading country in number of critical cases and the third in number of new deaths caused by COVID-19, while São Paulo State accounts for more than 33% of total con rmed cases in Brazil. Therefore, understanding the mechanisms of SARS-CoV-2 spread in São Paulo State is an important task. The aim of this study was to assess environmental and social factors in uencing the spread of SARS-CoV-2 in the expanded metropolitan area of São Paulo, Brazil. Firstly, a spatial analysis was conducted to provide insights into the spread of COVID-19 within the expanded metropolitan area. Moreover, statistical analyses were performed to assess social indicators and environmental conditions which possibly in uence the incidence of COVID-19. Our results reveal that the spread of COVID-19 from the capital city São Paulo-its epicenter in Brazil-is directly associated with the availability of highways within the expanded metropolitan area of São Paulo. As for social aspects, cumulative COVID-19 con rmed cases were found to be both positively correlated with population density, and negatively correlated with social isolation rate, hence, indicating that social distancing has been effective in reducing the COVID-19 transmission. Finally, cumulative COVID-19 con rmed cases were found to be inversely correlated with both temperature and UV radiation. Together with recent literature our study suggests that the UV radiation provided by sunlight might reduce the infectivity of SARS-CoV-2.

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In vivo fluorescence imaging in the near-infrared region between 1500–1700 nm (NIR-IIb window) affords high spatial resolution, deep-tissue penetration, and diminished auto-fluorescence due to the suppressed scattering of long-wavelength photons and large fluorophore Stokes shifts. However, very few NIR-IIb fluorescent probes exist currently. Here, we report the synthesis of a down-conversion luminescent rare-earth nanocrystal with cerium doping (Er/Ce co-doped NaYbF4 nanocrystal core with an inert NaYF4 shell). Ce doping is found to suppress the up-conversion pathway while boosting down-conversion by ~9-fold to produce bright 1550 nm luminescence under 980 nm excitation. Optimization of the inert shell coating surrounding the core and hydrophilic surface functionalization minimize the luminescence quenching effect by water. The resulting biocompatible, bright 1550 nm emitting nanoparticles enable fast in vivo imaging of blood vasculature in the mouse brain and hindlimb in the NIR-IIb window with short exposure time of 20 ms for rare-earth based probes.

This protocol provides a method for quantitating the intracellular concentrations of endogenous metabolites in cultured cells. The cells are grown in stable isotope-labeled media to near-complete isotopic enrichment and then extracted in organic solvent containing unlabeled internal standards in known concentrations. The ratio of endogenous metabolite to internal standard in the extract is determined using mass spectrometry (MS). The product of this ratio and the unlabeled standard amount equals the amount of endogenous metabolite present in the cells. The cellular concentration of the metabolite can then be calculated on the basis of intracellular volume of the extracted cells. The protocol is exemplified using Escherichia coli and primary human fibroblasts fed uniformly with 13 C-labeled carbon sources, with detection of 13 C-assimilation by liquid chromatographytandem MS. It enables absolute quantitation of several dozen metabolites over ~1 week of work.

We followed 68 cellular metabolites after carbon or nitrogen starvation of Escherichia coli and Saccharomyces cerevisiae, using a filter-culture methodology that allows exponential growth, nondisruptive nutrient removal, and fast quenching of metabolism. Dynamic concentration changes were measured by liquid chromatography-tandem mass spectrometry and viewed in clustered heat-map format. The major metabolic responses anticipated from metabolite-specific experiments in the literature were observed as well as a number of novel responses. When the data were analyzed by singular value decomposition, two dominant characteristic vectors were found, one corresponding to a generic starvation response and another to a nutrient-specific starvation response that is similar in both organisms. Together these captured a remarkable 72% of the metabolite concentration changes in the full data set. The responses described by the generic starvation response vector (42%) included, for example, depletion of most biosynthetic intermediates. The nutrient-specific vector (30%) included key responses such as increased phosphoenolpyruvate signaling glucose deprivation and increased ␣-ketoglutarate signaling ammonia deprivation. Metabolic similarity across organisms extends from the covalent reaction network of metabolism to include many elements of metabolome response to nutrient deprivation as well.Escherichia coli ͉ metabolomics ͉ nitrogen/carbon metabolism ͉ Saccharomyces cerevisiae ͉ starvation response T he pathways of cellular metabolism are close to identical across widely divergent organisms (1). The prokaryote Escherichia coli and the eukaryote Saccharomyces cerevisiae share essentially the same metabolic network (2) despite radically different compartmentation. The concentrations and fluxes of metabolites depend on the interactions among this conserved network structure, the cellular environment, and species-specific factors, such as the location, activities, and regulation of metabolic enzymes. Except for a few classic examples, e.g., central carbon metabolism in E. coli and yeast (3-5) and nitrogen assimilation in bacteria (6-8) the effect of environmental nutrient perturbations on the cellular metabolome have not been directly measured.We explored exponentially growing E. coli and S. cerevisiae cultures suddenly deprived of their carbon or nitrogen sources. Metabolic composition can change in as little as a few seconds (9, 10). To get accurate snapshots of the metabolome, we devised a method of growing cells directly on filters to avoid time-consuming procedures (e.g., centrifugation or filtration) before quenching of biochemical activity (11). Transfer of a filter from a plate containing growth medium into cold organic solvent quickly quenches metabolism; transfer to a plate with a different medium composition allows a quick change of the nutrient environment (Fig. 1).For measurement of a number of metabolites simultaneously, we used a liquid chromatography-electrospray ionization-triple quadrupole mass spectrometry (LC-MS/...

Despite extensive study of individual enzymes and their organization into pathways, the means by which enzyme networks control metabolite concentrations and fluxes in cells remains incompletely understood. Here, we examine the integrated regulation of central nitrogen metabolism in Escherichia coli through metabolomics and ordinary-differential-equation-based modeling. Metabolome changes triggered by modulating extracellular ammonium centered around two key intermediates in nitrogen assimilation, a-ketoglutarate and glutamine. Many other compounds retained concentration homeostasis, indicating isolation of concentration changes within a subset of the metabolome closely linked to the nutrient perturbation. In contrast to the view that saturated enzymes are insensitive to substrate concentration, competition for the active sites of saturated enzymes was found to be a key determinant of enzyme fluxes. Combined with covalent modification reactions controlling glutamine synthetase activity, such active-site competition was sufficient to explain and predict the complex dynamic response patterns of central nitrogen metabolites.

This protocol enables quantitation of metabolic fluxes in cultured cells. Measurements are based on the kinetics of cellular incorporation of stable isotope from nutrient into downstream metabolites. At multiple time points, after cells are rapidly switched from unlabeled to isotope-labeled nutrient, metabolism is quenched, metabolites are extracted and the extract is analyzed by chromatographymass spectrometry. Resulting plots of unlabeled compound versus time follow variants of exponential decay, with the flux equal to the decay rate multiplied by the intracellular metabolite concentration. Because labeling is typically fast (t 1/2 ≤5 min for central metabolites in Escherichia coli), variations on this approach can effectively probe dynamically changing metabolic fluxes. This protocol is exemplified using E. coli and nitrogen labeling, for which quantitative flux data for ~15 metabolites can be obtained over 3 d of work. Applications to adherent mammalian cells are also discussed.

Highlights d Impaired alveolar regeneration results in sustained elevated tension on alveoli d Elevated mechanical tension activates a TGF-b signaling loop in AT2 cells d The impacts of mechanical tension on alveoli are not uniformly distributed d Mechanical tension-activated TGF-b signaling is essential for fibrosis progression